isolation and characterization of cytolytic protein gene
TRANSCRIPT
Isolation and Characterization of Cytolytic Protein
Gene from Local Isolates of Bacillus thuringiensis
By
Rabia Faiz
32-GCU-PHD-Z-11
DEPARTMENT OF ZOOLOGY
GC UNIVERSITY LAHORE i
Isolation and Characterization of Cytolytic Protein
Gene from Local Isolates of Bacillus thuringiensis
Submitted to GC University Lahore
in partial fulfillment of the requirements
for the award of degree of
Doctor of Philosophy
IN
Zoology
By
Rabia Faiz
32-GCU-PHD-Z-11
DEPARTMENT OF ZOOLOGY
GC UNIVERSITY LAHORE ii
DECLARATION
I, Ms. Rabia Faiz Registration No. 32-GCU-PHD-Z-11 hereby declare
that the matter printed in the thesis titled “Isolation and characterization of
cytolytic protein gene from local isolates of Bacillus thuringiensis” is my
own work and has not been submitted and shall not be submitted in future as
research work, thesis for the award of similar degree in any University,
Research Institution etc in Pakistan or abroad.
At any time, if my statement is found to be incorrect, even after my
Graduation, the University has the right to withdraw my PhD. Degree.
Dated: ____________
_______________________
Signatures of Deponent
iii
PLAGIARISM UNDERTAKING
I, Ms. Rabia Faiz Registration No. 32-GCU-PHD-Z-11 solemnly declare
that the research work presented in the thesis titled “Isolation and
characterization of cytolytic protein gene from local isolates of Bacillus
thuringiensis” is solely my research work, with no significant contribution
from any other person. Small contribution/ help wherever taken has been
acknowledged and that complete thesis has been written by me.
I understand the zero tolerance policy of HEC and Government College
University Lahore, towards plagiarism. Therefore I as an author of the above
titled thesis declare that no portion of my thesis has been plagiarized and any
material used as reference has been properly referred/ cited.
I understand that if I am found guilty of any formal plagiarism in the
above titled thesis, even after the award of PhD. degree, the University
reserves the right to withdraw my PhD. degree and that HEC/ University has
the right to publish my name on HEC/ University website, in the list of culprits
of plagiarism.
Dated: ____________
_______________________
Signatures of Deponent
iv
RESEARCH COMPLETION CERTIFICATE
Certified that the research work contained in this thesis titled
“Isolation and characterization of cytolytic protein gene from local
isolates of Bacillus thuringiensis” has been carried out and completed by
Ms. Rabia Faiz Registration No. 32-GCU-PHD-Z-11under my supervision.
This thesis has been submitted in partial fulfillment of the requirements for the
award of degree of Doctor of Philosophy in Zoology.
Date Supervisor
Submitted Through
Dr. Muhammad Tahir ________________________
Chairperson Controller of Examinations
GC University Lahore. Department of Zoology
GC University Lahore.
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Acknowledgements
All praises and thanks are for almighty ALLAH Who is the most Gracious and Merciful. He
is the most compassionate and beneficent, Who consecrated mankind with the knowledge
and ability to think into his secrets. He bestows me the sense of knowledge and enables me to
accomplish this task.
All respects to Holy Prophet Muhammad (PBUH), who directed the humanity and
mankind from the world of darkness to an era of peace and enlightened the hearts with
Almighty Allah’s message.
I would like to express my deep and sincere gratitude to my supervisor, Dr. Dilara Abbas
Bukhari, Associate Professor, whose encouragement, guidance and full support from initial
to final level enabled me to develop a better understanding of my research work. Her support
and motivation has been very helpful during the course of this research project.
I would like to express my heartily thanks to Assoc. Prof. Dr. Muhammad Tahir, Chairman
Department of Zoology, Government College University, Lahore for encouragement and
sincere guidance regarding my work.
I also warmly thank to respected, Prof. Dr. Azizullah, for his kind cooperation, valuable
advices and guidance during the research project.
My heartiest thanks to Dr. Tayyab and Dr. Nadeem, University of The Punjab. I am also
thankful to Dr. Muhammad Tariq Zahid for his help during research work.
I am also thankful to Imran Haider Bhai, Asghar Bhai, Wadood Bhai, Umar Draz, Adnan
Saleem, Inam Bhai, Ali Imran and all the junior staff for their help and corporation.
I would like to thank my research fellows, Ms. Safia Rahman, Ms Bushra Kaleem and Mr
Sajjad and Ms. Nousheen for their friendly help in my research work.
I owe my heartiest thanks to my loving and caring husband Rahat Rauf and my sweet, lovely
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kids Aashir and Haania, without their cooperation, encouragement, compromises and
understanding it would not have been possible for me to complete my PhD.
And in the last but not least this would not have been possible without the prayers of
my Parents Mr & Mrs Dr. Faiz Ahmad and family especially my younger sister Ammara
Faiz who really supported me alot especially during my late working hours and also my in-
laws especially my mother in-law. May Allah Pak bless them all.
Rabia Faiz
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Dedication
Dedicated to my loving and caring
husband my sweet, cute lovely kids
Aashir and Haania and of course to
my loving Parents.
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List of Contents
Contents Page No
1. Introduction 1
Bacillus thuringiensis (B.t.) 2
History of Bacillus thuringiensis (B.t.) 3
Habitat of Bacillus thuringiensis (B.t.) 4
Insecticidal proteins of Bacillus B.t. 4
Classification of insecticidal proteins of B.t. 5
Cry proteins 6
Cyt proteins 6
Cytolytic proteins 11
Classification of Cyt proteins 12
Structure of Cyt toxins 14
Mode of action of Cyt toxins 15
Mechanism of membrane insertion 16
Pore formation model 16
Detergent effect model 16
Cyt 1 proteins 17
Cyt 1A 17
Cyt 1Aa 17
Cyt 1Ca 18
Cyt 2 proteins 19
Cyt 2 A 19
Cyt 2Aa2 19
Cyt 2B proteins 23
Cyt 2Bb1 23
Resistance to B.t. 23
Synergism between Cyt and Cry toxins 25
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Contents Page No
2.Material and methods 29
Sample collection 29
Isolation of bacteria 30
Morphological characterization 31
Colony morphology 31
Gram’s staining 31
Endo spore staining 31
Biochemical tests for the identification of B.t. 32
Motility test 32
Catalase test 32
Indole test 32
Starch hydrolysis test 32
Casein hydrolysis test 33
Gelatin hydrolysis test 33
Citrate utilization test 33
Voges- Proskauer test 33
Tyrosine utilization test 33
Growth on Sabouraud dextrose agar 33
Growth on 7% NaCl 34
Intracellular protein crystal production 34
PCR based detection of cyt gene 34
Primers used 34
Conditions for PCR 34
Screening of most toxic B.t. isolates positive for cyt gene 35
Larval Bioassays using B.t. spores 35
Bacterial spore dose preparation 35
Determination of spore concentration 36
Spore counting 36
Experimental setup for Bioassays 36
Larval Bioassays using total B.t.cell protein 36
Extraction of B.t. cell protein 36
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Estimation of total protein content 37
Bradford method 37
Lowry method 37
Experimental setup for Bioassays 37
Molecular characterization of most toxic B.t. isolates 37
PCR amplification of 16S rRNA gene 38
Primers used 38
Conditions for PCR 38
Growth characteristics of selected B.t. isolates 39
Determination of optimum growth temperature 39
Determination of optimum pH 39
Determination of optimum inoculum size 39
Determination of growth curve 39
Determination of antibiotic sensitivity and resistance 40
Determination of protein profile of selected B.t. isolates 40
Protein isolation method 1 40
Protein isolation method 2 41
Sodium dodycyl sulphate polyacrylamide gel electrophoresis (SDS-
PAGE) 41
Cloning of full length cyt 2B gene 41
Amplification of full length cyt 2B gene 41
Primers used 41
PCR conditions 42
Purification of PCR product of cyt 2B gene 42
Cloning of full length gene 43
Ligation of cyt 2B gene in pTZ57R/T 43
Prepration of competent cells 43
Transformation of competent cells with recombinant
plasmid 43
Selection of transformed recombinant colonies 45
Isolation of recombinant plasmid by alkaline lysis method 45
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Contents Page No
Restriction analysis of recombinant plasmid 45
Expression of full length cyt 2B gene in E.coli 46
Construction of recombinant DNA 46
Transformation of competent cells 46
Screening of positive clones 46
Expression of recombinant protein 47
Isolation of recombinant protein 48
Analysis of expressed recombinant protein 48
Optimization of conditions for the expression of recombinant
protein 48
Purification of expressed Cyt 2B protein 49
High alkaline pH stress 49
An ion exchange chromatography 49
Biotoxicity assay with expressed Cyt 2b protein against Aedes
aegypti larvae 49
Bioassay with E.coli transformed with cyt 2B gene 49
Bioassay with total expressed proteins of E.coli
transformed with cyt 2B gene 50
Bioassay with purified expressed cyt 2B protein 50
Results 51
B.t. isolates 51
Characteristics of B.t. isolates 51
Prevalence of cyt 2B gene in the B.t. isolates 52
Characteristics of cyt 2B positive B.t. isolates 55
Colony morphology 55
Microscopic observations 55
Gram’s staining 57
Endo spore staining 57
Antibiotic resistance of B.t. isolates 57
Biotoxicity assay with B.t. isolates 59
Bioassays with B.t. spores 59
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Contents Page No
Bioassays with total B.t. cell proteins 65
Comparison of LC 50 of spores and total cell proteins 65
Sequencing of shorter fragment of cyt 2B gene 65
Ribotyping of B.t. isolates 69
Growth characteristics of selected B.t. isolates 76
Optimum growth temperature 76
Optimum pH for growth 77
Optimum inoculum size 77
Growth curve of B.t. isolates 77
Molecular characterization of full length cyt 2B gene 77
Amplification of full length cyt 2B gene 77
Cloning and sequencing of full length cyt 2B gene 77
Expression of full length cyt 2B gene in E.coli 84
Optimum conditions for the expression of full length
Cyt 2B protein 84
Deduced amino acid sequence of cyt 2 B gene of
GCU Bt4 84
Composition of cyt 2B gene of Bt isolates 85
Purification of expressed Cyt 2B protein 85
An ion exchange chromatography of partially purified Cyt
2B protein 87
3D structure of Cyt 2B protein from NBBt4 87
Bioassays with expressed Cyt 2B protein 89
Discussion 91
References 98
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List of Figures
Figures Page No
Figure: 1.1. Phylogram demonstrating amino acid sequence
identity among Cry and Cyt proteins. 13
Figure: 1.2. Phylogram showing relationships between Cyt
family components. 14
Figure: 1.3.Three-dimensional structure of cyt toxins from
Bacillus thuringiensis. 15
Figure: 1.4. Mechanisam of action of cyt 2Aa2 toxin protein. 19
Figure: 2.1. Diagrammatic representation of PCR reaction
cycle for the amplification of shorter fragment of cyt 2B gene. 35
Figure: 2.2. Diagrammatic representation of PCR reaction cycle
for the amplification of 16SrRNA gene. 38
Figure: 2.3. Diagrammatic representation of PCR reaction
cycle for the amplification of full length cyt 2B gene. 42
Figure: 2.4. Map of cloning vector pTZ57R/T. 44
Figure: 2.5. Map of Expression vector pET 22b. 47
Figure: 3.1. Agarose gel electrophoresis of PCR product of
469 bp fragment of cyt 2B gene. 55
Figure: 3. 2. (A) Frequencies of B.t. isolates in different soil samples,
(B) Frequencies of cyt 2B positive B.t. isolates in different soil
samples. 56
Figure: 3.3. Colony morphology of B.t. isolate positivr for cyt
2B gene. 57
Figure: 3.4. Gram’s staining (A) and endospore (B) staining
of GCU B.t. 4. 59
Figure: 3.5. Mortality (%) caused by spores of six most toxic B.t. isolates
against 3rd
instar larvae of Ae. aegypti. 62
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Figures Page No
Figure: 3.6. Mortality (%) caused by total cell proteins of six most toxic
B.t. isolates against 3rd
instar larvae of Ae. aegypti. 67
Figure: 3.7. Comparison of LC50 of B.t. spores (Brown) and total B.t.
cell proteins (maroon) against 3rd
instar larvae of Ae. aegypti. 68
Figure: 3.8. Phylogenetic relationship of shorter fragment of cyt
2B gene from the most toxic B.t. isolate NBBt4 with already
reported cyt 2B genes. 69
Figure 3.9. Phylogenetic relationship of shorter fragment of cyt 2B gene
from the six most toxic B.t. isolate NBBt1-6 with each other. 69
Figure 3.10. Gel electrophoresis of 16SrRNA gene of six most toxic
B.t. isolates. 70
Figure 3.11. Phylogenetic relationship of the 6 most toxic B.t. isolates
with each other and with already reported B.t. strains. 76
Figure: 3.12. Effect of temperature on the growth of B.t. isolates. 78
Figure: 3.13. Effect of pH on the growth of B.t. isolates. 79
Figure: 3.14. Effect of inoculum size on the growth of B.t. isolates. 80
Figure: 3.15 (A&B). Gel electrophoresis of full length cyt 2B gene. 81
Figure; 3.16. Fig 3.16 (A), (B) Agarose gel electrophoresis of full
length cyt 2B gene. 82
Figure: 3.17. Restriction digestion of pTZ57R/T with EcoRI and
HindIII 83
Figure: 3.18. Restriction digestion of pET22b with NdeI and
BamHI 83
Figure: 3.19. Protein profile of B.t. isolates 84
Figure: 3.20. Protein profile of E. coli transformed with cyt 2 B gene
after IPTG induction 85
Figure: 3.21. Purification of partially purified Cyt 2B prtein of NBBt 4
after anion exchange chromatography 87
Figure: 3.22. Homology based 3D structure of Cyt 2B protein of
NBBt4 generated through EXPACY TRANSLATE TOOLS 88
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List of Tables
Tables Page No
Table: 1.1. Known cry and cyt gene sequences with revised nomenclature assignments.
6
Table: 1.2. The STs associated with major B.t.serovers. 12
Table: 1.3. cyt2-positive strains: host ranges and the presence
of IS240 region. 20
Table: 1.4. cyt2-negative strains: host ranges and the presence of IS240- related sequences
21
Table: 1.5. Toxicities of B. sphaericus, Cyt1Ab, Cyt2Ba, and
combinations of B. sphaericus and Cyt1Ab or Cyt2Ba toward
susceptible (strain Syn-P) and B. sphaericus-resistant (strain Bs-R)
C. quinquefasciatus. 27
Table: 2.1. List of sampling sites from where soil samples were
collected along with pH and temperature of the soil. 29
Table: 3.1. Gram staining, Endospore staining and biochemical
characterization of local B.t. like isolates. 52
Table: 3.2. List of B.t. isolates identified on the basis of staining and
biochemical tests along with sampling localities. B.t. isolates positive
for cyt 2B gene are also indicated. 53
Table: 3.3. Morphological characteristics of B.t. isolates positive for
cyt 2B gene. 58
Table: 3.4. Behaviour of B.t. isolates positive for cyt 2B gene
against different antibiotics. 60
Table: 3.5. Toxicity (% mortality) of sporulated forms of B.t. isolates
positive for cyt 2B gene, used at a concentration ranging between
100µg-1000 µg/ml against 3rd
instar larvae of Aedes agepti exposed
for 24 hours. 61
Table: 3.6. Toxicity (% mortality) of sporulated forms of B.t. isolates
positive for cyt 2B gene, used at a concentration ranging between
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Tables Page No
1100µg-2000 µg/ml against 3rd
instar larvae of Aedes agepti exposed
for 24 hours. 63
Table: 3.7. The six most toxic B.t. isolates, isolated from different
localities and soil along with soil texture, against 3rd
instar larvae of
mosquito Aedes agepti. 64
Table: 3.8. Toxicity (% mortality) of the total cell protein of six most
toxic B.t. isolates against 3rd
instar larvae of mosquito Aedes agepti. 66
Table: 3.9. Comparison of toxicity of spores and total cell protein of
B.t. isolates against 3rd
instar larvae of Aedes aegypti. 68
Table: 3.10. Amino acid composition (number and percentage) of cyt 2
B protein of six Bt isolates. 86
Table; 3.11. Different steps for purification and yield of cyt 2B protein
of GCU Bt4 88
Table; 3.12. Toxicity (%age mortality) of recombinant organism (E.coli BL21C
transformed with plasmid containing cyt 2B gene), expressed crude
protein and expessed and purified protein against Ae. aegypti larvae. 90
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Summary
Chemical insecticides are widely used to control the insects, these insecticides are not
environmentally healthy as they are not biodegradeable and hence are biomagnified. These
insecticides are also not host specific; they also kill the beneficial insects. So there is a need
to search for a control agent which should not harm the environment and also to human
beings. Several different methods have been used in recent past to control the insects which
include use of pheromones for trapping or disruption of mating behavior, insect growth
regulators that interfere with larval development, parasitoids, fungi, viruses and bacteria,
which debilitate or cause death in the infected insect.
One of the most successful biological control organisms is a naturally occurring
bacterial pathogen, Bacillus thuringiensis generally known as B.t. Formulations based on B.t.
have been used for decades as biological insecticides for agriculture and forestry, as well as
for vector control against mosquitoes and black flies. Interest in B.t. proteins has increased
during the last two decades because of their unique qualities which are unmatched by any
conventional insecticide. Of the 297 genes known to encode B.t. proteins, some share a high
degree of homology, while others have diverse nucleotide sequences. Because of the interest
in B.t., the list of new B.t. subspecies is growing as is the group of economically important
target insects. B.t. produces crystal proteins during sporulation. These crystal proteins are of
two types Cry and Cyt. Both of these types of proteins are different in their mode of action.
Cytolytic proteins have an additional property of having cytolytic activity against different
cells and also against mammalian erythrocytes. These proteins especially Cyt proteins are
active against mosquito larvae.
Recent studies reported the development of resistance in mosquitoes against
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Cry toxins. Researchers tried different methods to overcome this resistance and they found that
when Cyt proteins are used in combination with Cry proteins they greatly reduced the
resistance of mosquitoes against B.t. toxins. This indicated that Cyt proteins work
synergistically with Cry proteins.
In the present study, soil samples collected from different areas of Lahore, Kasur and
Faisalabad. A total of 50 soil samples were collected, these soils were rich in organic manure.
B.t. like bacteria were isolated from these soil samples using differential medium containing
sodium acetate buffer. These isolates were then subjected to biochemical characterization by
performing biochemical tests. The expected B.t. like isolates were screened for the presence
of cyt genes. After confirmation of presence of cyt 2B gene, mosquitocidal activity of these
isolates were checked by using B.t. spores and total B.t. cell proteins against 3rd
instar larvae.
From the bioassays, it was found that NB B.t.4 was found to be most toxic with LC50 value of
400±1.15 µg/ml and 68±0.46 µg/ml for its spores and total cell protein, respectively. After
bioassays, six most toxic B.t. isolates were then selected for further study.
Ribotyping of these isolates was done to amplifying 16S rRNA gene to identify these
isolates. Protein profile of these isolates was checked to confirm the presence of 29 kDa
protein band. Full length cyt 2B gene was amplified, cloned in pTZ57RT cloning vector, and
pET22b vector was used for expression. IPTG induction of 1 mM was found good for
expression ranging incubation time of 4-6 hours. Expressed protein was then purified by
anion exchange chromatography. Bioassays were performed using recombinant organism (E.
coli transformed with cyt 2B gene), expressed crude protein and purified protein. It was found
that the purified protein was most toxic with LC50 Value of 50±1.68 µg/ml.
1
INTRODUCTION
Chemical insecticides which are used to control the vectors of different
diseases spoil the environment as being creating bad and harmful impacts on humans
as well as natural environment. Insect pests and diseases resulting in crop damage
could cause up to 35% total loss (Pardo-Lopéz et al., 2013). During the last 25 years
mosquito species namely Anopheles gambiae (A. gambiae) and Culex pipiens (C.
pipiens) which are vectors of malaria and West Nile virus respectively developed
resistance to these insecticides especially in Europe, America and Africa. The
development of this resistance is probably due to the insensitivity of their
acetylcholinesterase to carbamates and organophosphates, components of the
commercial insecticides (Weill et al., 2003).
This kind of problems can be solved by using alternate methods to control
vectors and one of such method is the use of biological control (Federici et al., 2010)
with varying levels of suitability, diversity and adaptation (Shan et al., 2005).
Biological control methods being practiced successfully include the use of
pheromones for trapping or disruption of mating behavior, insect growth regulators
that interfere with larval development, parasitoids, fungi, viruses and bacteria, which
debilitate or cause death in the infected insect (Way and van Emden, 2000).
One of the most successful biological control organisms is a naturally
occurring bacterial pathogen, Bacillus thuringiensis (generally known as ―B.t.‖).
Formulations based on B.t. have been used for decades as biological insecticides for
agriculture and forestry, as well as for vector control against mosquitoes and black
flies (Becker and Margalit, 1993). Interest in B.t. proteins has increased during the
last two decades because of their unique qualities which are unmatched by any
conventional insecticide (Whalon and Wingerd, 2003). Of the 297 genes known to
encode B.t. proteins (Crickmore, 1998), share a high degree of homology, while
others have diverse nucleotide sequences. Moreover, the list of new B.t. subspecies is
growing as is the group of economically important target insects. The existence of a
large number of B.t. endotoxins is one of the advantages of using B.t. over most
synthetic chemical insecticides. The efficacy of existing endotoxins has improved
2
along with continued search for new and interesting sequences. Combining different
types of toxins in the formulation has also proven useful as a strategy for increasing
the host range and for delaying resistance build-up against these toxins (Marrone and
Macintosh, 1993).
Although much progress has been made in the 100 years since B.t. was first
identified, the search for new genes and the potential of existing genes has not been
exhausted. Many B.t. strains previously identified as toxic for one kind of insect are
effective for other kinds as well. Moreover, chimeric expression of these cry genes
has in some cases, interesting toxic effects on target and non-target insects (Schnepf
et al., 1998).
1.1. Bacillus thuringiensis (B.t.)
Bacillus thuringiensis (B.t.) is a Gram positive (Schneph et al., 1998) spore
forming bacterium that produces crystal proteins (Li et al., 1996) during sporulation
phase, which are environment friendly, insecticidal in nature and are also called Cry
and Cyt toxin (Schneph et al., 1998; Bravo et al., 2007; Bravo et al., 2011). These
crystalline bodies consist of protein subunits called δ-endotoxins. These para sporal
and crystal-associated features distinguish B.t. toxins from other toxins. Furthermore,
the proteinaceous toxins produced by B.t. are the active insecticidal agents. However,
they are harmless to human beings, birds, and vertebrates, as well as to beneficial
insects and to plants. B.t. proteins possess a highly specific insecticidal activity, with
different strains of the B.t. exhibiting different insect-host spectra. Although several
toxins may be associated with a particular isolate of B.t., some isolates produce only
one toxin. The genes coding for both these toxins are present on the plasmid therefore
the genes are called plasmid borne (Hofte and Whiteley, 1989). Both these toxins
belong to a group of bacterial toxins commonly famous as pore forming toxins
(PFTs). PFTs are the proteins which can only be inserted into their host membrane
through pores when these undergo conformational changes, and destroy host cells by
disrupting their ion balance (Park et al., 2005).
There are thousands of different B.t. strains, producing over 200 Cry proteins
that are active against an extensive range of insects and some other invertebrates (Li
et al., 1996). These Cry toxins are active against Dipterans, Coleopterans,
Lepidopteran insects (Misztal et al., 2004), Hymenoptera and Nematodes (Bravo et
al.,2007) have also been used for the control of mosquitoes and several crop pests
3
while Cyt toxins are specifically active against Dipteran insects, mosquitoes and
black flies (Chilcott and Ellar, 1988; Koni and Ellar, 1994; Orduz et al., 1996; de
Maagd et al., 2003) and some Cyt toxin are also found active against coleopteran
larvae like Cyt1Aa, found toxic against Chrysomela scripta, Cyt 2Ca, found toxic
against Diabrotica spp. and Leptinotarsa decemlineata (Soberon et al., 2012). The
Cyt toxins produced by B.t. not only exhibit insecticidal activity but also exhibited
cytolytic activity invitro against different cell lines and these toxins also showed
hemolytic activity (Thomas et al., 1983; de Maagd et al., 2003). The mosquitocidal
activity of Cyt toxins alone is however lower than that of the Cry toxins (Crickmore
et al., 1995; Orduz et al., 1996).
1.1.1. History of Bacillus thuringiensis (B.t.)
It was a setback for the world of fashion, but a golden opportunity for the
world of agriculture when a Japanese biologist, Shigetane Ishiwatari, investigated the
cause of the sotto disease (sudden-collapse disease) in 1901. The disease was killing
large populations of silkworms when Ishiwatari first isolated a bacterium as the
disease- causing agent (Nester et al., 2002). Silkworm larvae suffering from
―flacherie‖ were found to be infected by a rod-shaped, gram-positive, spore-forming
bacterium. Ishiwatari named the bacterium Bacillus sotto but the name was later ruled
invalid. In 1911, Ernst Berliner isolated a bacterium that had killed a Mediterranean
flour moth, and rediscovered Bacillus sotto. He named it Bacillus thuringiensis (B.t.),
after the German town Thuringia where the moth was found. Berliner reported the
existence of a crystal within B.t. in 1915, but the activity of this crystal was not
discovered until much later (Kreig, 1986). In 1955, researchers, Hannay and Fitz-
James reported that the main insecticidal activity against Lepidopteran (moth) insects
was due to the parasporal crystal. This discovery increased interest in the crystal
structure, biochemistry, and general mode of action of B.t.
The organism has been isolated from many insects and shows indications of
being an opportunistic pathogen in the insect larvae (Schnepf et al., 1998). B.t. first
became available as a commercial insecticide in France in 1938. In the 1950s it
entered commercial use in the United States. For many years, B.t. primarily came in
the form of a spray to be applied to crops (van Frankenhuyzen, 1994). The earliest
report of a commercial B.t. preparation was in 1938 and the product was called
"Sporeine" (Jacobs, 1951). This product is still being used in experiments to control
4
the flour moth Ephestia kuehmella z. In 1951, Steinhaus is credited with raising the
profile of B.t. as a biological control alternative in the USA. His efforts resulted in the
commercialization in 1957 of the product "Thinicide" (Biofenn Corp.), based on B.t.
var. thuringiensis. In the meantime, the first international standard for evaluating the
potency of B.t. commercial products was established in 1966 (Burges, 1967).
New markets were opened by the discovery in 1976 of the israelensis
subspecies, which is toxic to larval mosquitoes and black flies (Margalit and Dean,
1985) and the discovery of the tenebrionis subspecies, which is toxic to several beetle
species. In recent years, there has been tremendous renewed interest in B.t. and
several new products have been developed, largely because of the safely associated
with B.t. based insecticides. Today, there are thousands of known strains of B.t. of
which many have genes in their DNA that encode unique toxic crystals. The B.t. as a
microbial insecticide was originally registered in 1961 as a general use insecticide.
The genes that encode the toxic crystals were moved into a plant with the
advancement in plant transgenic technology. The first genetically engineered plant,
corn, was registered with the EPA in 1995 (Betz et al., 2000). This wealth of strains
and toxins will surely keep increasing as the emerging field of genomics offers its
analysis and harvest.
1.1.2. Habitat of B.t.
B.t. strains have been isolated from diverse habitats such as, soil and aquatic
environments (Ichimatsu et al., 2000), plants (Maduell et al., 2002), insects
(Cavadoes et al., 2001), animal faeces (Lee et al., 2003) and arid environments
(Assaeedi et al., 2011) including beaches, desert, and tundra habitats (Li et al., 1996),
and also from forest land soil (Yu et al., 2015). The natural occurrence of B.t. in the
environment and its insecticidal activity has been reported from various countries in
Europe (Apaydin et al., 2008), North America, South America (Park et al., 2008),
Asia (Gao et al., 2008) and Africa (Ogunijimi et al., 2000). In the Middle East, the
natural occurrence of B.t. in soil environments was reported from Egypt (Merdan and
Labib, 2003) and Jordan (Sadder et al., 2006).
1.1.3. Insecticidal proteins of B.t.
B.t. produces insecticidal proteins at two different stages during its growth.
One type of proteins that B.t. produces very early during the vegetative stage. These
proteins are called vegetative insecticidal proteins (Vip).
5
Other types of proteins constitute two families of δ-endotoxins (Estruch et al., 1996)
which are produced later during sporulation phase as para sporal crystals and are
called as crystal (Cry) and cytolytic (Cyt) proteins (Hofte and Whiteley, 1989).
1.1.3.1. Classification of insecticidal proteins of B.t.
B.t. is unique in its ability to produce crystalline inclusions during sporulation.
For a long time these crystalline inclusions have caused great controversy in the
classification of different bacteria. Heimpel and Angus (I960) concluded that
previous attempts to establish a taxonomic status for the crystal-forming bacteria
proved confusing. They proposed a classification based on similarities with the
Bacillus cereus group while providing a new species designation for the B.t. Their
criteria were based on traditional biochemical and morphological characteristics. A
year later Toumanoff and Le Corroller (1959) proposed a scheme in which the
―crystalliferous‖ group was differentiated, in some instances, based on the host from
which the organism was isolated. They recognized different characteristics depending
on the host and suggested that B.t. remain within the B. cereus group. Heimpel in
1967 presented yet another taxonomic key that proposed variety distinction based on
toxicity.
H-antigen serotyping was an approach proposed by De Barjac and Bonnefoi
(1968) and De Barjac et al., (1988). The different approaches that have been
proffered in an attempt to better describe these bacteria have resulted in references to
serovars, biovars and crystovars. Since the insecticidal activities of these bacteria
were the main concern of the pathologist, the classifications based on crystovars and
biovars proved most useful at the bench. Much of this early confusion has been
explained by the discovery that the genes coding for the crystal protein are often
found on plasmids. Gonzales et al. (1981), have shown that these conjugative
plasmids can be lost or expressed depending on culture conditions. In 1989, many of
these genes had been sequenced, forming the basis of a classification system by
crystal gene type (Hofie and Whiteley, 1989).
The criteria for different gene types were based in part, on the specificity
towards certain orders (Lepidoptera, Coleoptera or Diptera), and on the comparison
of sequence homologies. This system relied on the insecticidal activities of crystal
proteins for the primary ranking of their corresponding genes. According to this
classification, cry I genes encode proteins toxic to Lepidopterans; cry II genes encode
6
proteins toxic to both Lepidopterans and Dipterans; cry III genes encode proteins
toxic to Coleopterans; and cry IV genes encode proteins toxic to Dipterans alone.
This system provided a useful framework for classifying the ever-expanding set of
known genes. However, inconsistencies existed in the original scheme due to
attempts to accommodate genes that were highly homologous known genes but did
not encode a toxin with a similar insecticidal spectrum. To circumvent these
problems, Crickmore et al. (1998), proposed a nomenclature for B.t. cry and cyt
genes.
1.1.3.1.1. Cry proteins: According to this system the definition of a Cry protein is
rather broad: a para sporal inclusion (crystal) protein from B.t. that exhibits some
experimentally verifiable toxic effect on a target organism, or any protein that has
obvious sequence similarity to a known Cry protein.
1.1.3.1.2. Cyt proteins: The complete list of all cry and cyt genes renamed by this
system is shown in Table 1.1 (Crickmore et al., 1998). Cyt denotes a para sporal
inclusion (crystal) protein from B.t. that exhibits hemolytic activity, or any protein
Table1.1. Known cry and cyt gene Sequences With revised
nomenclature assignments
Revised gene Original gene or protein Accession Coding
name Name no. region
a
cry1Aa1 cryIA(a) M11250 527–4054
cry1Aa2 cryIA(a) M10917 153–>2955
cry1Aa3 cryIA(a) D00348 73–3600
cry1Aa4 cryIA(a) X13535 1–3528
cry1Aa5 cryIA(a) D17518 81–3608
cry1Aa6 cryIA(a) U43605 1–>1860
cry1Ab1 cryIA(b) M13898 142–3606
cry1Ab2 cryIA(b) M12661 155–3622
cry1Ab3 cryIA(b) M15271 156–3620
cry1Ab4 cryIA(b) D00117 163–3627
cry1Ab5 cryIA(b) X04698 141–3605
cry1Ab6 cryIA(b) M37263 73–3537
7
Revised gene Original gene or protein Accession Coding
name Name no. region
a
cry1Ab7 cryIA(b) X13233 1–3465
cry1Ab8 cryIA(b) M16463 157–3621
cry1Ab9 cryIA(b) X54939 73–3537
cry1Ab10 cryIA(b) A29125 — b
cry1Ac1 cryIA(c) M11068 388–3921
cry1Ac2 cryIA(c) M35524 239–3769
cry1Ac3 cryIA(c) X54159 339–>2192
cry1Ac4 cryIA(c) M73249 1–3534
cry1Ac5 cryIA(c) M73248 1–3531
cry1Ac6 cryIA(c) U43606 1–>1821
cry1Ac7 cryIA(c) U87793 976–4509
cry1Ac8 cryIA(c) U87397 153–3686
cry1Ac9 cryIA(c) U89872 388–3921
cry1Ac10 AJ002514 388–3921
cry1Ad1 cryIA(c) M73250 1–3537
cry1Ae1 cryIA(e) M65252 81–3623
cry1Af1 Icp U82003 172–>2905
cry1Ba1 cryIB X06711 1–3684
cry1Ba2 X95704 186–3869
cry1Bb1 ET5 L32020 67–3753
cry1Bc1 cryIB(c) Z46442 141–3839
cry1Bd1 cryE1 U70726
cry1Ca1 Cryic X07518 47–3613
cry1Ca2 Cryic X13620 241–>2711
cry1Ca3 Cryic M73251 1–3570
cry1Ca4 Cryic A27642 234–3800
cry1Ca5 Cryic X96682 1–>2268
8
Revised gene Original gene or protein Accession Coding
name Name no. region
a
cry1Ca6 Cryic X96683 1–>2268
cry1Ca7 Cryic X96684 1–>2268
cry1Cb1 cryIC(b) M97880 296–3823
cry1Da1 cryID X54160 264–3758
cry1Db1 prtB Z22511 241–3720
cry1Ea1 cryIE X53985 130–3642
cry1Ea2 cryIE X56144 1–3513
cry1Ea3 cryIE M73252 1–3513
cry1Ea4 U94323 388–3900
cry1Eb1 cryIE(b) M73253 1–3522
cry1Fa1 cryIF M63897 478–3999
cry1Fa2 cryIF M73254 1–3525
cry1Fb1 prtD Z22512 483–4004
cry1Ga1 prtA Z22510 67–3564
cry1Ga2 cryIM Y09326 692–4210
cry1Gb1 cryH2 U70725
cry1Ha1 prtC Z22513 530–4045
cry1Hb1 U35780 728–4195
cry1Ia1 cryV X62821 355–2511
cry1Ia2 cryV M98544 1–2157
cry1Ia3 cryV L36338 279–2435
cry1Ia4 cryV L49391 61–2217
cry1Ia5 cryV159 Y08920 524–2680
cry1Ib1 cryV465 U07642 237–2393
cry1Ja1 ET4 L32019 99–3519
cry1Jb1 ET1 U31527 177–3686
cry1Ka1 U28801 451–4098
9
Revised gene Original gene or protein Accession Coding
name Name no. region
a
cry2Aa1 cryIIA M31738 156–2054
cry2Aa2 cryIIA M23723 1840–3738
cry2Aa3 D86064 2007–3911
cry2Ab1 cryIIB M23724 1–1899
cry2Ab2 cryIIB X55416 874–2775
cry2Ac1 Cryic X57252 2125–3990
cry3Aa1 cryIIIA M22472 25–1956
cry3Aa2 cryIIIA J02978 241–2172
cry3Aa3 cryIIIA Y00420 566–2497
cry3Aa4 cryIIIA M30503 201–2132
cry3Aa5 cryIIIA M37207 569–2500
cry3Aa6 cryIIIA U10985 569–2500
cry3Ba1 cryIIIB2 X17123 25–>1977
cry3Ba2 cryIIIB A07234 342–2297
cry3Bb1 cryIIIBb M89794 202–2157
cry3Bb2 cryIIIC(b) U31633 144–2099
cry3Ca1 cryIIID X59797 232–2178
cry4Aa1 cryIVA Y00423 1–3540
cry4Aa2 cryIVA D00248 393–3935
cry4Ba1 cryIVB X07423 157–3564
cry4Ba2 cryIVB X07082 151–3558
cry4Ba3 cryIVB M20242 526–3930
cry4Ba4 cryIVB D00247 461–3865
cry5Aa1 cryVA(a) L07025 1–>4155
cry5Ab1 cryVA(b) L07026 1–>3867
cry5Ac1 I34543 1–>3660
cry5Ba1 PS86Q3 U19725 1–>3735
10
Revised gene Original gene or protein Accession Coding
name Name no. region
a
cry6Aa1 cryVIA L07022 1–>1425
cry6Ba1 cryVIB L07024 1–>1185
cry7Aa1 cryIIIC M64478 184–3597
cry7Ab1 cryIIIC(b) U04367 1–>3414
cry7Ab2 cryIIIC(c) U04368 1–>3414
cry8Aa1 cryIIIE U04364 1–>3471
cry8Ba1 cryIIIG U04365 1–>3507
cry8Ca1 cryIIIF U04366 1–3447
cry9Aa1 cryIG X58120 5807–9274
cry9Aa2 cryIG X58534 385–>3837
cry9Ba1 cryX X75019 26–3488
cry9Ca1 cryIH Z37527 2096–5569
cry9Da1 N141 D85560 47–3553
cry9Da2 AF042733 <1–>1937
cry10Aa1 cryIVC M12662 941–2965
cry11Aa1 cryIVD M31737 41–1969
cry11Aa2 cryIVD M22860 <1–235
cry11Ba1 Jeg80 X86902 64–2238
cry11Bb1 94 kDa AF017416
cry12Aa1 cryVB L07027 1–>3771
cry13Aa1 cryVC L07023 1–2409
cry14Aa1 cryVD U13955 1–3558
cry15Aa1 34kDa M76442 1036–2055
cry16Aa1 cbm71 X94146 158–1996
cry17Aa1 cbm72 X99478 12–1865
cry18Aa1 cryBP1 X99049 743–2860
cry19Aa1 Jeg65 Y07603 719–2662
11
Revised gene Original gene or protein Accession Coding
name Name no. region a
cry19Ba1 D88381
cry20Aa1 86kDa U82518 60–2318
cry21Aa1 I32932 1–3501
cry22Aa1 I34547 1–2169
cyt1Aa1 cytA X03182 140–886
cyt1Aa2 cytA X04338 509–1255
cyt1Aa3 cytA Y00135 36–782
cyt1Aa4 cytA M35968 67–813
cyt1Ab1 cytM X98793 28–777
cyt1Ba1 U37196 1–795
cyt2Aa1 cytB Z14147 270–1046
cyt2Ba1 “cytB” U52043 287–655
cyt2Bb1 U82519 416–1204
aThe symbols < and > indicate that the coding region extends up- or downstream, respectively, from the known sequence data.
bOnly the polypeptide sequence has been reported.
that has obvious sequence similarity to a known Cyt protein. Some of the B.t. strains
that exhibit biopesticidal activity contains distinct sequence types called STs that they
do not share with any strain of B. cereus group (Raymond and Federici., 2017), (Table
1.2)
1.1.3.2. Cytolytic proteins
The cyt genes corresponding to hemolytic toxins constitute a large and diverse
gene family just like the larvicidal δ-endotoxins the cry genes in B.t. The presence of
Cyt hemolytic factor in B.t. is specifically associated with mosquitocidal strains of B.t.
and recently it has also been shown that different patho types also contain these cyt
genes. This is especially the case of B.t. subsp. morrisoni and some other stains active
against Coleopteran, Lepidopteran and Dipterans. It was also observed that in most
toxic anti-dipteran strains at least two Cyt toxins coexist such as in B.t. subsp.
israelensis and subsp. morrisoni PG14 (Guerchicoff et al., 2001).
12
Table 1.2. The STs associated with the major B.t. serovars used in insect pest
management are all recovered from insect and environmental sources. Unique
sequence ST numbers are defined here according to unique allele profiles in the
MLST scheme developed by Priest et al., 2004.
Product
names
Bt
serovar Isolate synonyms ST
Isolates with identical
allele profile in
SuperCAT (and
pub.mlst) databases
DiPel BMP
123 Thuricide kurstaki HD-1 8 79 (74)
XenTari,
Florbac, aizawai T07033/HD227 15
a 8 (7)
Novodor Morrisoni BGSC4AA1 biovar.
Tenebrionis 23 23 (21)
Tekar,
VectoBac,
Aquabac
israelensis BGSC4Q1,ONR60A, H-
14, ATCC 35 646 16
b 6
Tekar,
VectoBac, israelensis BGSC4Q7 HD1002 16 21 (13)
aNot confirmed: other aizawai STs include 53, 54, 833, 834.
bClosest match based on available allelic profile: gmk 7; ilv7; pta 2; pur 6; pyc 8; tpi 13.
1.1.3.2. Classification of Cyt proteins
Fig 1.2. shows relationship between different Cyt proteins based on full length
gene sequences (Crickmore et al., 1998). Different species of B.t. produce cytolytic
proteins with very little variations in their amino acid sequences. Cyt proteins are
classified into three classes according to their amino acid sequence identity i.e., Cyt1,
Cyt2 and Cyt 3 (Crickmore et al., 2015).
13
Fig 1.1. Phylogram demonstrating amino acid sequence identity among Cry and Cyt
proteins. This phylogenetic tree is modified from a TREEVIEW visualization of
NEIGHBOR treatment of a CLUSTAL W multiple alignment and distance matrix of
the full-length toxin sequences,
14
Fig. 1.2: Phylogram showing relationships between Cyt family components. Thicker
vertical lines demarcate the four levels of nomenclature ranks according to the results of
Crickmore et al.,1998. Protein names in boldface indicate that the central regions of the
genes have been used for comparison.
1.1.3.3. Structure of Cyt toxins
According to Guerchicoff et al. (2001) and Crickmore et al. (1998), more than
thirty Cyt toxins have been identified so far. Cyt toxins share high homology in their
structure as indicated by their amino acid sequence. The three Cyt proteins namely
Cyt 1Aa, Cyt 2Aa and Cyt 2Ba share almost a similar structure as depicted by the X-
ray crystallographic analysis, having two layers of outer α-helix hairpins flanking
around a core of β-sheets representing a cytolysin fold in a single domain structure
(Li et al., 1996; Cohen et al., 2008; Cohen et al., 2011.) (Fig.1.3). This structure show
striking similarity with fungal volvatoxin A2, Erwinia virulence factor (Evf) and it
may be predicted that both of these proteins may have a similar mode of action
against insect cell membrane (Lin et al., 2004; Cohen et al., 2008.).
The hydrophobic residues of α-helices are packed against the β-sheet
exhibiting amphipathic character of α-helices (Thomas and Eller, 1983). In order to
span the membrane lipid bi layer, the α-helices of these Cyt toxins are not long
15
Fig. 1.3: Three-dimensional structure of Cyt toxins from Bacillus thuringiensis
displayed by Swiss PDB viewer. Cyt 1Aa, pdb 3RON; Cyt2Aa, pdb 1CBY; and
Cyt2Ba, pdb 2RCI.
enough for this purpose. Unlike α-helices, β-sheet has a length that it could easily
span the cell membrane of host (Li et al., 1996).
1.1.3.4. Mode of action of Cyt toxins
Cyt toxins show insecticidal activity in vivo, causes hemolysis of red blood
corpuscles and cytolytic activity to different cultured cells in vitro (de Maagd et al.,
2003; Thomas and Ellar, 1983). The crystalline endotoxins are predominantly
synthesized as inactive protoxins. Upon activation, they are highly toxic to their target
hosts. When insects digest the crystals, the alkaline digestive juice in the midgut
solubilizes them. Once solubilized, endotoxins are activated by proteases, interacting
with the midgut epithelial cells of susceptible insects. The specificity of B.t.
endotoxins towards particular insects implies the presence of specific receptors in the
target tissue. The binding and insertion of the toxin in the midgut leads to the
formation of a pore or lesion in the plasma membrane followed by cell lysis,
disruption of gut integrity and, eventually, death of the insect from starvation or
septicemia (Aronson and Shai, 2001).
The Cyt 1Aa toxin of the mosquitocidal B.t. var. israelensis possesses
different receptor recognition and binding mechanism. No specific receptors have
been described for Cyt toxins, although they show binding specificity invivo (Schnepf
et al., 1998). Initially, Cyt A binds to unsaturated phospholipids (Thomas and Ellar,
1983;Gill et al., 1987) and aggregates of about 300-400kDa are formed, leading to
pore formation and cytolysis (Chow et al., 1989).
B.t. synthesizes Cyt proteins as inactive toxins or pro toxins which are
converted into active toxins of 25 kDa by mid gut proteases in a process called as
proteolytic activation. Binding of activated Cyt toxins to the mid gut epithelium of
16
susceptible insect does not require midgut proteases rather these toxins bind to the
mid gut epithelium by interacting with unsaturated membrane lipids such as
sphingomyelin, phosphatidylcholine and phosphatidylethanolamine (Ward and Ellar,
1983).
1.1.3.4.1. Mechanism of membrane insertion
As for as the mechanism of action of Cyt toxins is concerned two models have
been proposed for membrane insertion of these toxins into the midgut of Dipteran
insects.
1.1.3.4.1.1. Pore formation model
First model is the pore formation model and according to this model the
binding of Cyt toxin to the membrane of mid gut cells induces the formation of cation
selective channels there. The formation of these channels basically occurs in the
membrane vesicles that ultimately lead to colloid osmotic lysis of cells (Knowles et
al., 1989, 1992; Promdonkoy and Ellar, 2003).
During the process of pore formation the two α-helix hair pins lying outside to
the β-strands swing apart from β-strands allowing β-strands to insert into the
membrane. A structured β-barrel pore is formed due to oligomerization within the
membrane that ultimately leads to colloid osmotic lysis of cells (Promdonkoy and
Ellar, 2000).
1.1.3.4.1.2. Detergent effect model
Second model is the detergent effect model according to which membrane of
the mid gut cells disassembles that ultimately results in cell death, due to the
nonspecific aggregation of toxins on the surface of membrane lipid bilayer (Butko,
2003; Manceva et al., 2004).
According to this model the aggregated Cyt toxins are absorbed on the
membrane surface resulting in defects in lipid packing (Butko, 2003; Manceva et al.,
2005). It is very likely similar to the carpet model which was formulated for
antimicrobial peptides (Shai, 1995). According to this model, the proteins may be
unstructured rather than the insertion into membrane. It was shown that the toxin
attains a conformation very likely to resemble with molten-globule state upon binding
of Cyt 1Aa to lipids (Manceva et al., 2004). It was thus suggested that disorder occur
in membrane lipids in the presence of Cyt toxin resulting in membrane break down
into lipid-protein complexes (Butko, 2003; Manceva et al., 2005).
Both of the models are not clashing or conflicting, rather each of these models
can operate or work at different time scales or they can operate at different toxin
17
concentrations. At higher concentrations of toxin the lipid membrane may be unable
to accommodate or manage a large number of toxin molecules that leads to
membrane breakup while at lower concentrations of toxin oligomeric pores may be
formed (Butko, 2003).
1.1.3.5. Cyt 1 proteins
1.1.3.5.1. Cyt 1A
1.1.3.5.1.1. Cyt 1Aa
Toxicity of Cyt 1Aa based on structural features is correlated to its ability to
undergo conformational changes just before insertion into the membrane and
perforation (Cohen et al., 2008, 2011). Cytolysin fold of the toxin enables the α-
helices to swing away exposing β-sheet that can now be inserted into the membrane.
The hemolytic activity of Cyt 1Aa, between the β-sheets presence of lipid binding
pocket and the helical layer of Cyt 1Aa resembling with the saponin and the pore
forming agents like α-toxin support this mechanism (Cohen et al., 2011).
Studies indicated that binding of Cyt 1Aa to the membrane of epithelial cells
of Dipterans mid gut is probably due to the strong affinity of this toxin to the
unsaturated fatty acids that makeup the membrane and not because of the binding of
Cyt 1Aa to specific receptors (Gill et al., 1987; Canton et al., 2014). A 22-25 kDa
fragment was obtained after in vitro processing of Cyt 1A protoxin (Al-yahyaee and
Ellar, 1995; Cahan et al., 2008) and as compared to protoxin this activated toxin is
three times more effective (Butko et al., 1996, 1997).
Cyt 1Aa binds to the stomach cells, brush border of mid gut cells and to the
gastric caeca of larvae of A. gambiae, this binding is may be the due to the ability of
toxin to penetrate the cell membrane without involvement of any receptor
(Ravoahangimalala and Charles, 1995). The higher affinity of Cyt toxins towards
Dipteran cell membrane and also the in vivo activity may be the result of high
concentration of unsaturated phospholipids in Dipteran insects than in any other
group of insects. This indicates a specific mode of action that is different from that of
B.t.i Cry toxins (Koni and Ellar, 1993; Li et al., 1996).
Cyt 1A97, a mutated gene of Cyt 1Aa, was isolated from B.t.i. strain
BUPM97. Nucleotide sequence revealed a 249 amino acid protein with 27 kDa
molecular mass. Nucleotide and amino acid sequence analysis revealed that there is
only one amino acid difference between cyt 1A97 and cyt 1Aa gene. It was observed
that this mutation is located on α-helix corresponding to substitution of Met at
position 115 with a Thr. The expression of mutated cyt 1A97 was performed in E.
coli and a cyt 1Aa type gene, named cyt 1A98 served as a control being not affected
18
by such mutation. It was observed that cyt 1A97 was over expressed in E. coli as
inclusion bodies resulting in very low toxicity to E. coli as compared to cyt 1A98 that
stopped growth of E. coli. This may be the result of Met at position 115, a
hydrophobic residue of cyt 1Aa playing an important role in the maintenance of
structure and cytolytic properties of cyt 1Aa, that was lacking in the mutated gene cyt
1A97 (Zghal et al., 2008).
The mosquitocidal strains of B.t. subspecies israelensis (B.t.i) have been
observed to have synergistic effect of Cyt and Cry toxins (Chang et al., 1993; Wu D
et al., 1994). B.t.i produces parasporal crystals of two types namely Cry and Cyt
proteins. Cry proteins include Cry 4Aa, Cry Ba, Cry 10Aa and Cry 11Aa while Cyt
proteins include Cyt 1Aa and Cyt 2Ba (Berry et al., 2002). The individual toxicities
of these toxins are lower but the combined effect of their toxicity against mosquito
larvae is higher than it was expected on the basis of their individual toxicities
(Crickmore et al., 1995; Perez et al., 2005).
1.1.3.5.2. Cyt 1Ca
Berry et al., (2002) reported a new 60-kDa Cyt like protein named Cyt 1Ca.
Structure of Cyt 1Ca comprises of a two domain fusion protein. The N-terminal part
of this fusion protein resembles to the full length Cyt toxins and its C-terminal part
resembles the receptor binding domains of Mtx1 which is a ricin- like toxin. It was
observed that Cyt 1Ca expressed in E. coli does not appear to have larvicidal activity
nor hemolytic activity of His-tagged purified Cyt 1Ca was observed (Manasherob et
al., 2001). This difference in toxicity may be due to the difference in five amino acids
in N-terminal of Cyt 1Ca and that of Cyt 1Aa. It was also observed that the 3 end of
Cyt 1 Ca was pruned resulting in the removal of ricin-binding domain, and sight
directed mutagenesis resulting in six single base changes that sequentially replaced
non-charged amino acids with charged ones. The expression of this mutated Cyt 1Ca
protein results in antimicrobial activity causing loss of colony forming ability of the
corresponding E. coli in which it is expressed. This antimicrobial effect of mutated
Cyt 1Ca against E. coli reflects an evolutionary relationship between Cyt 1Ca and Cyt
1Aa (Itsko et al., 2005).
1.1.3.6. Cyt 2 protein
It was observed that all the B.t. strains with known anti Dipteran activity were
found to be positive for cyt 2 related genes. Protein sequence alignment suggested
high degree conservation in the structural domains. This structural conservation
seems to play an important role in the invivo toxicity of B.t. strains (Guerchicoff et
al., 2001).
19
1.1.3.6.1. Cyt 2 A
1.1.3.6.1.1. Cyt 2Aa2
Tharad et al. (2015) used two different techniques; the atomic force
microscopy (AFM) and quartz crystal microbalance with dissipation (QCM-D) in
order to investigate the binding mechanism and interaction of Cyt 2Aa2 protein. They
found that the binding properties of Cyt 2Aa2 depend upon the concentration of
protein used. They observed that at low protein concentration i.e., 10 µg/ml Cyt 2Aa2
binds slowly to the lipid bilayer resulting in the formation of a concurrence protein/
lipid layer with aggregates, while at higher concentrations i.e., 100 µg/ml the binding
of Cyt 2Aa2 is faster resulting in more rigid protein/ lipid layer including holes in its
structure (Fig. 1.4).
Fig. 1.4: Mechanisam of action of Cyt 2Aa2 toxin protein (Tharad et al., 2015). x
Table 1.3. cyt2-positive strains: host ranges and the presence of IS240 region.
Strain Subspecies Serotype
Host PCR IS240
c
range a
bands b
1884 Israelensis H14 1 469 +
4Q2-72 Israelensis H14 1 469 +
PG14 Morrisoni H8a, H8b 1, 3 469 +
B51 AAT021 ND 1 469 +
11S2-1 Canadensis H5a, H5c 1 469 +
B175 Thompsoni H12 1 469 +
IMR 81-1 Malaysiensis H36 1 469 +
CIB 163-131 Medellin H30 1 469 +
367 Jegathesan H28a, 1 469 +
20
H28c
74-F-6-18 Kyushuensis H11a,
1
469
+ H11c
84-I-1-13 Kukuokaensis H3a, H3d,
1
469
+ H3e
73-E-10-2 darmstadiensis H10a,
1
469
+ H10b
B.006 Ostriniae H8a, H8c ND 469 +
78-FS-29-17 Tohokuensis H17 ND 469 +
Serovar Morrisoni H8a, H8b 2
469, 600 −
Tenebrionis
HD12 Morrisoni H8a, H8b 1, 3 469 +
HD518 Morrisoni H8a, H8b 1, 3 469 +
EA10192 andalousiensis H37 4 469 +
aHost range was scored as follows: 1, toxic for dipteran larvae; 2, toxic for coleopteran larvae; 3,
toxic for lepidopteran larvae; and 4, nontoxic for dipteran larvae (Culex or Aedes sp.).
bOccurrence of cyt2-related genes based on PCR amplification with cyt2B-specific primers (in base
pairs). cOccurrence of IS240 sequences based on hybridization with a IS240A probe (35).
dND, not
determined.
Table 1.4. cyt2-negative strains: host ranges and the presence of IS240-
related sequences
` Strain Subspecies Serotype Host range
a IS240
b
T08001 Morrisoni H8a, H8b 4 +
DD960 Thompsoni H12 4 +
T03C001 Fukuokaensis H3a, H3d, H3e 1 −
GM33 Monterrey H28a, H28b 4 +
271 Colmeri H21 4 +
Indiana Indiana H16 ND +
Yunnanensis Yunnanensis H20a, H20b ND +
273B Cameroun H32 4 +
Finitimus Finitimus H2 4 +
HL51 Leesis H33 1* +
Pak 94 Pakistani H13 4 +
21
3-71 kumamotoensis H18a, H18b 4*
+
Ygd22-03 Jinghongiensis H42 4* +
HL47 Konkukian H34 4*
+
19-105 Novosibirsk H24a, H24c ND +
GM18 Neoleonensis H24a, H24b ND +
Berliner Thuringiensis H1 3 −
SLM 5.A Silo H26 4* +
92-KU-137-4 Higo H44 4** +
Sotto G Sotto H4a, H4b 3 +
HL1 Coreanensis H25 4 +
GM43 Mexicanensis H27 4*
−
LFB 855 Oswaldocruzi H38 4*
+
ABr33 Londrina H10a, H10c 4 +
H11 Toumanoffi H11a, H11b 1* +
B23 pondicheriensis H20a, H20c ND +
` Strain Subspecies Serotype Host range a
IS240 b
Toguchini Toguchini H31 4*
+
DMU-38 Roskildiensis H45 4 +
KK31-01 Guiyangiensis H43 4 +
T2 shandongiensis H22 ND +
8 4-F-58.20 Amagiensis H29 4*
+
aHost range was scored as follows: 1, toxic for dipteran larvae (*, weakly mosquitocidal for Culex
sp.); 2, toxic for coleopteran larvae; 3, toxic for lepidopteran larvae; 4, nontoxic for dipteran larvae (*,
Culex or Aedes sp.; **, moderate antidipteran activity). ND, not determined.
bOccurrence of IS240 sequences based on hybridization with a IS240A probe.
Promdonkoy et al., (2003) reported PCR amplification using specific primers
designed from cyt 2 Aa1 gene sequences of B.t. subsp. kyushuensis. Analysis of DNA
sequence indicated an open reading frame translating a sequence of 259 amino acids.
This gene was designated as cyt 2 Aa2. In E. coli this gene is highly expressed as
inclusion bodies. These inclusion bodies were solubilized using sodium carbonate
buffer pH 10.5. Solubilized pro toxin was activated by using 1% proteinase K
22
yielding a 23 kDa active fragment. This activated fragment of Cyt 2Aa2 was found
toxic against Ae. agypti and C. quinquefasciatus larvae and also showed hemolytic
activity against sheep erythrocytes.
B.t. subsp. darmstadiensis produces cytolytic toxin Cyt 2Aa2 that exhibits
cytolytic activity against broad range of cells in vitro and in vivo toxicity against
Dipteran larvae. In order to determine the role of amino acids in αA and αC of this
toxin protein 3 single point mutations were generated at A61C, S108C ANS V109A.
These three mutant proteins were highly expressed as inclusion bodies. These
inclusion bodies were then solubilized and activated by using proteinase K just like
wild type protein. It was observed that the hemolytic activity of S108C and A61C
mutated proteins was reduced significantly while V109A mutated protein showed a
comparable hemolytic activity to that of wild type. It was also observed that the
larvicidal activity of A61C mutated protein was high against both C. quinquefasciatus
and Ae. aegypti while that of S108C and V109A mutated proteins exhibited low
larvicidal activity against C. quinquefasciatus and relatively high toxicity against Ae.
aegypti (Promdonkoy et al., 2008).
1.1.3.6.2. Cyt 2B
Mosquitocidal strains of B.t. exhibit toxin proteins with hemolytic and
cytolytic activities. B.t.i. contains a cytolytic protein Cyt 1Aa1, and another cytolytic
toxin very much similar to cyt 2Aa1 coding for a 29-kDa protein toxin has been
detected. This gene designated as cyt 2Ba1 present upstream of cry 4B gene encoding
130-kDa protein on a 72-MDa plasmid. PCR based amplification and Southern blot
analysis confirmed the presence of cyt 2B gene in other mosquitocidal subspecies of
B.t. (Guerchicoff et al., 1997).
Interestingly B.t. subsp. tenebrionis belonging to serover morrisoni, which is
active against Coleopteran larvae showed the presence of this gene, while negative
results were obtained for B.t. subsp. kurstaki HD-1 which is active against
Lepidoptera larvae and subsp. aizawai HD-137 (Guerchicoff et al., 1997).
1.1.3.6.2.1. Cyt 2Bb1
Cheong and Gill (1997) used limited-growth PCR screening method in order
to amplify a cytolytic toxin gene cyt 2Bb1 encoding 30.1kDa toxin protein from B.t.
subsp. jegathesan. Primers for this study were designed using N-terminal amino acid
sequence of native and trypsin activated protein. The expressed protein when treated
with antibodies raised against Cyt 1Aa protein showed very little cross reactivity. It
was also observed that this expressed protein produced little or no crystal inclusions
which very unlikely to Cyt 1Aa and Cyt 2Aa that produced crystal inclusions. A
23
sequence homology of 31% and 66% was observed to Cyt 1Aa and Cyt 2Aa when
amino acid sequences of this expressed protein were aligned. Sequence alignment of
five different known cytolytic proteins indicated three highly conserved regions, two
were present in the loop region between the α-helices and β-sheets, and the third one
was present in the loop region between β-sheets 5 and 6. β-blocks of this proteins are
also structurally conserved namely β-blocks 4-7. Bioassays indicated that this protein
has low mosquitocidal activity than Cyt 1Aa and 600 time low toxicity than wild type
whole Cry toxins. Cyt 2Bb and Cyt 1Aa showed almost similar hemolytic activity.
1.1.4. Resistance to B.t.
B.t. toxins cover a wide target range of insect pests, but the target insects may
develop resistance against the corresponding toxin at one stage or another (Zhu et al.,
2009). Incorporation of B.t. technology into an integrated pest management is the
preferred strategy to achieve effective insect control while minimizing target
resistance (Andow et al., 2001).
In 1985, the first report on insect resistance to B.t. was published (Mc
Gaughey, 1985) otherwise; many investigators mistakenly believed that insects would
not develop resistance to B.t. Since then, many more such cases have been reported
including other orders of insects. It now appears that resistance to B.t. can evolve
rapidly under situations of extreme selection pressure, whether in the laboratory or
the field (Stone et al., 1989; Tabashnik et al., 1990).
McGaughey (1985) studied the first case of resistance to B.t. in Indian meal
moth (Plodia interpunctel). Several generations of meal moth larvae were exposed to
high levels of Dipel (a commercial B.t. product based on B.t. var. kurstaki. Btk) and
eventually resistance was developed 100-fold. Resistance reached 250-fold after 36
generations of selection.
Resistance to B.t. in the diamondback moth has been reported in populations
in Hawaii, Florida, New York, China, Japan, Malaysia, the Philippines, and Thailand
(Tabashnik et al., 1990; Ferre et al., 1991; Tanaka and Kimura, 1991; Sayyed et al.,
2000; Sayyed et al., 2001). The diamondback moth (Plutella xylostella) is the only
insect species that has evolved high levels of resistance to B.t. in the field. The first
case of field resistance was reported from Hawaii. Populations from areas heavily
treated with Dipel proved less susceptible than populations that had been treated at
lower levels, with the highest level of resistance at 30-fold (Tabashnik et al., 1990).
Laboratory selection, however, using Dipel increased resistance drastically to over
1000-fold 9 (Tabashnik et al., 1991, 1994). A reversal of resistance from extremely
high levels (2800-fold) in P. xylostella occurred very rapidly in the absence of
24
selection pressure although susceptibility was not fully restored even after 39
generations (Tabashnik et al., 1994).
Several reports of cross-resistance in insects have been documented for B.t.
toxins. In 1998, Ramachandran et al., fed cry 1 A-resistant larvae of P. xylostella with
cry l Ac transgenic canola leaves. The larvae developed equally well on both
transgenic and normal leaves.
Colorado potato beetle (Leptinotarsa ilecem/ineata), established from adults
collected from fields sprayed with B.t. formulations containing Cry3Aa, when
subjected to laboratory selection with Cry3Aa formulations for 29 generations,
resulted in 293-fold resistance (Rahardja and Whalon, 1995).
Cyt IA plays an important role in the lack of development of resistance in
mosquitoes towards B.t. var. israelensis (B.t.i). Indeed, there are no reported cases of
field or significant laboratory resistance to B.t.i (Wirth and Georgiou, 1997). In
selection experiments using individual toxins or combinations of individual toxins
from B.t.i on a laboratory colony of C. quinquefasciatus, the rate and final level of
resistance (at least at the LC95 level, concentration required to kill 95% of insects)
was inversely correlated with the number of constituent toxins in the selecting agent.
Whereas all selected lines displayed varying levels of resistance to all selecting agents
not containing Cyt lAa, none of the selected lines showed significant levels of
resistance to the Cry 4A/ Cry 4B/ Cry l lA/ Cyt l A mixture. These results suggest that
the presence of Cyt lAa suppresses the development of resistance toward, Cry 4/ Cry
11 toxins. It is interesting that combining Cyt lAa with Cry 4 or Cry 11 proteins
restored the toxicity of Cry4 and Cry l I against C. quinquefasciatus colonies resistant
against the latter proteins (Wirth and Georghiou, 1997).
1.1.6. Synergism between Cyt and Cry toxins
Bacillus sphaericus strain 2362 produces binary toxins which are active
against Culex sp, has recently being developed as commercial larvicide. In several
countries like France, Brazil, India and USA it has been used in operational mosquito
control programs. In France, Brazil and India field resistance to B. sphaericus has
been reported. In order to manage this resistance Cyt 1Aa protein of B.t.i. De Barjac
(subsp) was evaluated against highly resistant C. quinquefasciatus population. For
this purpose, B. sphaericus strain 2362 and B.t.i. strain producing only Cyt1A were
used in combination in a ratio of 10:1. This reduced reistance upto 30,000 folds and
resistance was completely suppressed when B. sphaericus was used in combination
with purified crystals of Cyt 1A in a ratio of 10:1. This synergism between B.
sphaericus and Cyt 1A is responsible for the marked reduction in resistance of Culex
25
populations. This provided an evidence that Cyt 1A may enhances the toxicity by
enhancing the binding and insertion of toxin into the microvilli membranes of
mosquitoes (Wirth et al., 2000).
Wirth et al. (2001) studied the effect of two cytolytic toxins Cyt 2Ba from B.t.
subsp. israelensis and Cyt 1Ab from B.t. subsp. medellin together in combination
with B. sphaericus against Ae. aegypti an insensitive species and against a resistant
strain of quinquefasciatus .They found that the mixtures of B. sphaericus with either
of the two cytolytic toxins were synergestic and the resistance of C. quinquefasciatus
to B. sphaericus was decreased from 17,000 to only 2 folds when a mixture of B.
sphaericus and Cyt 1Ab was used in a ratio of 3:1 (Table 1.5).
26
27
28
MATERIALS AND METHODS
2.1. Sample collection
Soil samples were collected from different areas of Lahore, Faisalabad, and Kasur.
For sampling sterilized spatula was used and soil from 1.5 to 2 cm below the surface is used
and saved in sterilized zipper bags with proper labeling (Table 2.1.). Samples were then
brought to laboratory and stored at 4ᵒC for processing.
Table 2.1. Sampling sites from where soil samples were collected along with pH and
temperature of the soil.
Sr .No Area/ Locality Texture of soil pH of Temperature
sample Sample (ᵒC)
1 Road side Johar town, Dry soil 8.1 37
Lahore
2 Garden soil Johar town, Moist soil 8.51 37
Lahore
3 Canal side Campus area Moist soil 8.05 36
4 Canal side near Mall road Moist sol 8.25 38
5 Pak Arab Society E block Dry soil with 8.81 36
organic manure
6 Pak Arab Society B block Dry flaky soil 7.6 37
7 5 Km from Ferozepur road Moist soil rich in 8.12 37
near Pak Arab organic manure
8 Coriander field near Pak Dry soil 6.90 37
Arab
9 Carrot field near Pak Arab Dry flaky soil 6.87 37
10 Reddish field near Pak Arab Moist soil 6.98 38
11 Lady finger field near Pak Dry flaky soil 6.81 38
Arab
12 Coly flower field near Pak Dry soil 6.88 38
Arab
13 Cattle rearing area near Pak Soil with Decaying 8.98 37
Arab cattle waste
14 Cattle grazing area near Pak Soil with dry cattle 9.11 37
Arab Waste
15 Country side Kasur Dry sandy soil 8.20 39
16 Cattle rearing area Kasur Dry flaky .soil with 8.23 39
cattle waste
17 Cattle grazing area Kasur Moist soil 8.11 38
18 Rice field Kasur Dry soil with rice 7.86 38
Straw
19 Agriculture field Kasur Moist soil with 7.99 39
decaying cattle
waste
29
Sr. No Area/ Locality Texture of soil pH of Temperature
sample Sample (ᵒC)
20 GT road near Kala Shah Dry soil 8.53 39
Kaku (KSK) ,Lahore
21 KSK rice fields Dry flaky soil 8.89 38
22 Cattle rearing area near KSK Dry soil with cattle 9.32 38
waste
23 Cattle grazing area near KSK Dry sandy soil 8.15 38
24 Coriander field KSK Dry soil with 9.22 39
organic manure
25 Garden soil KSK Moist soil with 9.21 39
decaying cattle
waste
26 Carrot fields KSK Moist soil 8.54 39
27 Coly flower field KSK Dry sandy soil 8.23 37
28 Cabbage field KSK Dry soil 7.99 38
29 Reddish field KSK Moist soil 8.20 38
30 Lady finger field KSK Moist soil 8.20 38
31 GT road near Faisalabad Dry sandy soil 7.60 39
32 Country side Faisalabad Dry flaky soil 6.90 39
33 UAF Faisalabad Dry flaky soil 7.79 39
34 GT road near GC, Faisalabad Dry soil 8.11 38
35 Jhang road, Faisalabad Moist soil 8.23 38
36 Cattle rearing area Dry soil with cattle 9.24 38
waste
37 Cattle grazing area Moist soil 8.80 36
38 Agriculture field Moist soil 8.22 36
39 Agriculture field with weeds Dry flaky soil 8.14 38
40 Cattle rearing area Dry soil rich in 8.11 37
organic manure
41 Riawind road, Lahore Dry soil 7.58 37
42 Cattle rearing area Dry soil with cattle 8.64 39
waste
43 Cattle grazing area Moist soil 9.22 39
44 Country side Dry soil 8.55 39
45 Coriander field Dry soil 8.32 38
46 Carrot field Moist soil 8.32 38
47 Lady finger field Dry soil with 8.21 36
animal waste
48 Rice field Moist soil 8.11 36
49 Rice field Dry soil with rice 8.01 38
straw
50 Coriander field Dry soil 8.55 38
2.2. Isolation of Bacteria
Bacteria were isolated according to method described by Martin and Travers (1989),
using sodium acetate selective media. One gram soil sample was added to 10 ml of LB
medium buffered with 0.3 M sodium acetate in a conical flask and incubated at 30ᵒC for 4
hours in shaking incubator at 150 rpm. The broth was filtered using syringe filter of 0.25 µm
pore size. Filtrate was then heat shocked at 80ᵒC for 10 minutes and 200 µl of each heat
30
shocked filtrate was then spreaded on T3 agar plates and incubated at 37ᵒC for 24-48 hours.
B.t. like colonies were then picked and streaked on LB agar plates in order to get pure culture.
2.3. Morphological characterization
Isolated bacterial strains were characterized morphologically
2.3.1. Colony morphology
Isolated bacterial strains were characterized by colony morphology which includes
colony margins, color, texture and elevation if present. In order to check the staining behavior
of isolates, gram’s staining and endospore staining were performed according to method
described by Holt et al., (1994).
2.3.2. Gram staining
For Gram staining 24 hours old bacterial cultures were used. Thin smears were made
using glass slide. Slides were then air dried and heat fixed so that bacteria may not rinse away
during staining procedure. Glass slides were then flooded with crystal violet and allowed to
stand for 1 hour. After that glass slides were washed with decolorizer by gently running the
decolorizer on slide until the decolorizer became clear. Then flood the slide with gram iodine
solution for one minute and rinse with decolorizer. Slide was then flooded with counter stain
safranin and allowed to stand for 45 minutes. More stain was added in order to prevent it
from drying, rinsed with decolorizer, air dried and saved for observation under microscope at
100X magnification.
2.3.3. Endospore staining
Three (3) days old culture was used for endospore staining. Thin smears were heat
fixed after air drying. Slide was flooded with malachite green solution for 40 minutes
subjected to continuous steaming. Slides were then slightly rinsed with autoclaved distilled
water. Slides were flooded with safranin for 10 minutes, again rinsed with autoclaved
distilled water, air dried and examined under microscope at 100X. Gram positive rods with
oval or round greenish in appearance endospores were further processed for biochemical
tests.
2.4. Biochemical tests for identification of B.t.
Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994) was followed for
the identification of Bacillus species. Log phase bacterial cultures were used in order to
perform following biochemical tests according to Brown et al., (2004), Cheesbrough (1993),
and Sneath (1986).
31
2.4.1. Motility test
For motility test 100 ml semi solid agar medium was prepared and autoclaved at
121ᵒC for 15minutes. Then 5 ml of this medium was poured in each of the test tube and
waited till solidification of the media. Sterilized inoculating needle was used for a top to
bottom single stab inoculation and incubated for 24 hours at 37ᵒC. Diffused and spreaded
growth around the streak line indicated positive motility test.
2.4.2. Catalase test
Catalase test was performed by using 3% solution of hydrogen peroxide. Three (3) ml
of this solution was taken in each of the test tubes. Bacterial colonies of 24 hours culture
were immersed in hydrogen peroxide solution separately by using sterile inoculating loop.
Positive catalase activity was indicated by the formation of bubbles within few minutes.
2.4.3. Indole test
Tryptophan supplemented medium was prepared and autoclaved. Then 5 ml of this
broth poured in each test tube and inoculated with 24 hours bacterial cultures and incubated
at 37ᵒC for 24 hours. After incubation period, 0.5 ml of freshly prepared Kovac’s reagent was
added slowly. Positive indole test was indicated by the development of red color in top layer.
2.4.4. Starch hydrolysis test
Starch agar medium was prepared and autoclaved and poured into Petri plates. After
inoculation plates were placed in incubator at 37ᵒC for 2 days. Plates were then gently
flooded with dilute iodine solution. Appearance of clear zone around the streaks and rest of
the plate as blue color indicated that starch is hydrolyzed.
2.4.5. Casein hydrolysis test
Milk agar was prepared by mixing autoclaved 0.2% agar medium after it has been
cooled and skimmed milk and poured in Petri plates. After inoculation plates were incubated
at 37ᵒC for 14 days. Clear zone around the streak lines indicated positive results.
2.4.6. Gelatin hydrolysis test
For this test gelatin broth medium was prepared and autoclaved. Then 5 ml of
medium was poured in each test tube and inoculated. Tubes were Incubated at 37ᵒC for 2
days and then placed on ice for 5 minutes. Persistence of liquid medium indicated positive
result.
2.4.7. Citrate utilization test
Simmons citrate agar medium was prepared autoclaved, and 5 ml was poured into
each test tube and slants were made. Test tubes were incubated at 37ᵒC after inoculation.
Appearance of blue color indicated positive result.
32
2.4.8. Voges-Proskauer test
Glucose peptone medium was prepared and autoclaved. Five (5) ml of the medium
was poured into each test tube and after inoculation incubated at 37ᵒC for 2 days. After 2
days, I ml KOH solution and 3 ml α-nephthol were added to the incubated samples. Positive
test result was indicated by the development of pink color after 2 to 5 minutes.
2.4.9. Tyrosine utilization test
For this test 100 ml tyrosine agar medium was prepared and autoclaved. Then 15-20
ml of medium was poured in each Petri plate, inoculated and incubated at 37ᵒC for 14 days.
Tyrosine utilization was indicated by the appearance of clear zones around the streaks.
2.4.10. Growth on Sabouraud dextrose agar
For this test, 100 ml of Sabouraud dextrose agar medium was prepared and
autoclaved, poured in Petri plates and inoculated. Inoculated Petri plates were then incubated
at 37ᵒC for one day. Appearance of growth in the medium indicated positive results.
2.4.11. Growth on 7% NaCl
For this test, 100 ml of 7% NaCl agar medium was prepared and autoclaved. Medium
was poured into Petri plates and after inoculation, incubated at 37ᵒC for one day. Positive
results were indicated by the appearance of growth on the medium.
2.4.12. Intracellular protein crystal production
GNB agar medium was prepared, autoclaved and poured into Petri plates. For
inoculation, isolated one day old bacterial colony was used and after inoculation incubated at
37ᵒC for 7 days. Protein crystals were then observed under microscope.
2.5. PCR based detection of cyt gene
PCR amplification of cyt gene was carried out in order to identify the specific B.t.
strains positive for cyt gene.
2.5.1. Primers used
For this purpose following primers reported by Guerchicoff et al., (2001) were
used.
Forward, 5'- AATACATTTCAAGGAGCTA-3' Tm: 50°C and
Reverse, 5'- TTTCATTTTAACTTCATATC-3' Tm: 48°C
For PCR amplification, crude DNA was used according to Carozzi et al., (1991)
(appendix). Strains were grown on LB agar medium and crude DNA obtained was used for
PCR amplification. The reaction mixture includes Taq buffer, MgCl2, dNTPs, Primer
forward, Primer reverse, DNA and ddH2O.
33
2.5.2. Conditions for PCR
Shorter fragment of cyt 2B gene was amplified in thermal cycler under following
conditions: first denaturation step for 3 min at 94ᵒC following 30 cycles, and each cycle with
denaturation for 45 seconds at 94ᵒC, annealing for 45 seconds at 45ᵒC and elongation for 1
min at 72ᵒC. At the end of 30 cycles, a final elongation step at 72ᵒC for 5 min and final
storage was done at 4ᵒC (Fig. 2.1). Amplified PCR products were run on 1% agarose gel.
Bands were cut from the gel and Fermentas gel elution kit (K 0513) was used for gene
purification.
Denaturation
94 ᵒC 94 ᵒC
Extension
_____________ 72 ᵒC 72 ᵒC
3 min 45 sec __________
45ᵒC 1 min 5 min
____________ 4ᵒC
45sec _____
Annealing ∞
Fig. 2.1: Diagrammatic representation of PCR reaction cycle for the
amplification of shorter fragment of cyt 2B gene.
In order to check the homology of amplified cyt gene with already sequenced cyt
genes in Gene Bank sequence database, the sequences of cyt gene of the B.t. strains were
aligned and blasted on NCBI nucleotide blast. The gene sequences were then deposited in the
NCBI database.
34
2.6. Screening of most toxic B.t isolates positive for cyt
gene
B.t strains positive for cyt 2B gene were screened for their toxicity against mosquito
larvae by means of larval bioassays. For larval bioassays both B.t. spores and total cell
proteins were used.
2.6.1. Larval bioassays using B.t. spores
2.6.1.1. Bacterial spore dose preparation
B.t. spore dose was prepared by using method described by Makino et al., (1994). LB
broth was inoculated with single isolated colony of B.t. and incubated at 37ᵒC for 24 hours.
T3 sporulation medium was then streaked with this inoculum and incubated at 30ᵒC for 3
days. Plates were then scraped off with the help of autoclaved distilled water and centrifuged.
Pellets were washed twice with autoclaved distilled water and incubated for 40 minutes at
37ᵒC with 10 ml of KCl sodium phosphate buffer and centrifuged. Two washes were given
with autoclaved distilled water and spore pellet was incubated with 10 ml of urea buffer
along with 25 mM of 2-mercaptoethanol at 37ᵒC for 30 minutes and then centrifuged. Five
washes were given with autoclaved distilled water and spore pellet was stored at 4ᵒC.
2.6.1.2. Determination of spore concentration
Method described by Cavados et al., (2005) was used in order to determine the spore
concentration in spore pellets the bacterial strains were grown in sporulation medium until
sporulation reached 90%. For each sample 0.5 mg of spore pellet was taken and oven dried at
70ᵒC for one day. Samples were then transferred to desiccator until dried. It was then
weighed up to fourth decimal and mean weight of biomass was determined and all these
experiments were run in triplicates.
2.6.1.3. Spore counting
Serial dilution of dried spores was used with a final concentration of 1 µg/ml by using
autoclaved distilled water. Heat shock was given to the samples in water bath at 80ᵒC for 12
minutes and plated on T3 agar medium. Incubated at 30ᵒC for 72 hours and number of spores
per µg was counted as number of colonies per plate.
2.6.1.4. Experimental setup for bioassays
Third instar larvae of Ae. aegypti were used kindly provided by Malarial research
center bird wood road Lahore. Ten different doses viz., 0, 100, 200, 300, 400, 500, 600, 700,
800, 900 and 1000 µg/ml of B.t. spores were prepared for each of the bacterial strain with a
total volume of 100 ml. These doses were prepared in wide mouthed plastic cups and 50 third
instar larvae were added in each of the eleven cups which were then covered with fine net.
35
Room temperature was maintained at 25ᵒC. Larval mortality, larvae which were knocked
down at the bottom of the cup were considered dead, was recorded after 24 hours.
Assessment of the toxicity was determined through log probit analysis (Finney, 1971).
2.6.2. Larval bioassays using total B.t. cell protein
2.6.2.1. Extraction of B.t. cell protein
B.t. cell pellets were obtained as mentioned above in spore diet preparation. Pellets
were then resuspended in alkaline buffer of high pH and incubated at 37ᵒC for 3 hours with
shaking. After centrifugation pellet was discarded and supernatant saved for further analysis.
2.6.2.2. Estimation of total protein content
Total B.t. cell proteins was estimated by two methods viz, Bradford and Lowry
method.
2.6.2.2.1. Bradford method (Bradford, 1976)
Phosphate buffer was used to dilute protein samples and phosphate buffer alone was
used as blank as well. To I ml of protein sample added 5 ml of protein assay dye reagent,
mixed well by inversion and left at room temperature for about 10 minutes. Absorbance was
then measured at 595 nm.
Protein concentration was determined using BSA standard curve (appendix).
2.6.2.2.2. Lowry method (Lowry et al., 1951)
To the 1 ml of protein sample 4.5 ml of Protein Assay Reagent 1 was added and
incubated at room temperature for 10 minutes. After incubation added 0.5 ml of Protein
Assay Reagent 2 and again incubated at room temperature for 30 minutes. Absorbance was
measured at 660 nm and protein concentration was determined by using BSA standard curve.
2.6.2.3. Experimental setup for bioassays
For this experiment, B.t. strains which were found to be most toxic after spore
bioassay were used. The solubilized pro toxin was converted into active toxin and this is done
by trypsin activation (Correa et al., 2012). Different doses of B.t. total cell proteins
(activated) were prepared ranging from 25 to 250 µg/ml in 50 ml of distilled water and 50
larvae were added to each cup. Larval mortality was noted after 24 hours. Assessment of the
toxicity was determined through log probit analysis (Finney, 1971).
2.7. Molecular characterization of most toxic B.t. isolates
Molecular characterization of most toxic B.t. strains was done in order to identify the
expected B.t. strains. Genomic DNA isolation of B.t. isolates was done using phenole
chloroform extraction method (appendix) according to Sambrook and Russel (2001). DNA
isolation was confirmed through agarose gel (1%) electrophoresis.
36
2.7.1. PCR amplification of 16S rRNA gene
PCR amplification was performed for the conserved region of 16S rRNA gene.
2.7.1.1. Primers used
Universal primers used for the amplification of 16S rRNA gene were as follows:
Forward primer: TGAAAACTGAACGAAACAAAC , Tm: 56°C
Reverse primer: CTCTCAAAACTGAACAAAACGAAA ,Tm: 64°C
2.7.1.2. Conditions for PCR
PCR was done according to Saiki et al., (1988). The reaction mixture includes Taq
buffer, MgCl2, dNTPs, Primer forward, Primer reverse, DNA and ddH2O. Full length gene
was amplified in thermal cycler under following conditions: first denaturation step for 5 min
at 94ᵒC following 30 cycles, and each cycle with denaturation for 2 min at 94ᵒC, annealing
for 1 min at 52ᵒC and elongation for 2 min at 72ᵒC. At the end of 30 cycles, a final elongation
step at 72ᵒC for 7 min and final storage was don at 4ᵒC (Fig. 2.2). Amplified PCR products
were run on 1% agarose gel. Bands were cut from the gel and Fermentas gel elution kit (K
0513) was used for gene purification.
Denaturation
94 ᵒC 94 ᵒC
Extension
__________ 72 ᵒC 72 ᵒC
5 min 2 min ________
52 ᵒC 2 min 7min
____________
1 min 4 ᵒC
_____
Annealig ∞
Fig. 2.2: Diagrammatic representation of PCR reaction cycle for the amplification of
16SrRNA gene.
In order to check the homology of isolated B.t. strains with already sequenced genes
in Gene Bank sequence database, the sequences of 16S rRNA gene sequence of isolated B.t.
strains were aligned and blasted. The gene sequences of isolated B.t. strains were then
deposited in the NCBI database.
37
2.8. Growth characteristics of selected B.t. isolates
B.t. isolates, which were found to be most toxic after larval bioassays were then
selected for the determination of optimum growth conditions.
2.8.1. Determination of optimum growth temperature
LB broth medium was used for this purpose. Log phase bacterial cultures of the most
toxic B.t. isolates were used to inoculate 100 ml of LB broth at a concentration of 100 µl in
five sets, each for a single B.t. isolate. Flasks were then incubated at different temperatures
i.e., 25 ᵒC, 30 ᵒC, 37 ᵒC, 40ᵒC and 60ᵒC. After 10 hours of incubation, bacterial growth was
determined in terms of absorbance at 600 nm wavelength using spectrophotometer.
2.8.2. Determination of optimum pH
LB broth medium was used for this purpose. To inoculate 100 ml of LB broth 100 µl
of log phase B.t. cultures were used in each of the six sets for each of the following pH i.e., 4,
5, 6, 7, 8 and 9. Flasks were then incubated at 37ᵒC for 10 hours. Bacterial growth was
determined in terms of absorbance at 600 nm wavelength using spectrophotometer.
2.8.3. Determination of optimum inoculum size
LB broth medium (100 ml) was used in five sets for each of the B.t. isolate. Log
phase cultures were used for inoculation at 1%, 2%, 3%, 4%, 5%, 6% ,7%, 8%, 9% and 10%
inoculum. Flasks were then incubated at 37ᵒC for 10 hours. Bacterial growth was determined
in terms of absorbance at 600 nm wavelength using spectrophotometer. All the experiments
were run in triplicates.
2.8.4. Determination of growth curve
For the determination of growth curve, 100 ml of LB broth medium was used in
triplicates. Inoculation was done using 1% log phase B.t. culture and flasks were incubated at
37ᵒC for 24 hours. After every hour flasks were taken out of the incubator and OD was taken
using spectrophotometer at 600 nm. Growth curves were prepared by plotting graph between
OD (along Y-axis) and incubation time (along X-axis).
2.9. Determination of antibiotic sensitivity and resistance
In order to determine the antibiotic sensitivity and resistance of the six most toxic B.t.
isolates, 24 hour old cultures were used. For this purpose LB agar plates were streaked with
24 hour old bacterial colonies. Different antibiotics used were,
Streptomycin (30µg), Ampicillin (30µg), Amoxilin (30µg), Tetracyclin (30µg) and
Erythromycin (30µg). These antibiotic discs were placed on streaked agar plates at intervals
and incubated at 37ᵒC for 24 hours. After 24 hours of incubation, antibiotic sensitivity of B.t.
isolates was checked in terms of presence or absence of zone of inhibition.
38
2.10. Determination of protein profile of B.t. isolates
Protein profile of most toxic B.t. isolates was determined by using two methods and
were analyzed on polyacrylamide gel electrophoresis.
2.10.1. Protein isolation method 1
Protein isolation was done by using method described by Sayyed et al., (2000). B.t.
isolates were grown in LB broth (100 ml) containing ampicillin. Medium was then incubated
at 29ᵒC for 72 hours. When 90% of the B.t. culture had sporulated, heat shock was given for
30 minutes at 70ᵒC. One litter of LB broth was then inoculated with one ml of this heat
shocked culture without adding any antibiotic and incubated for 72 hours in shaking
incubator at 30ᵒC. It was then centrifuged at 15000 rpm for 10 minutes, supernatant was
discarded and pellet was saved. Pellet was resuspended in 25% initial volume of NaCl EDTA
buffer and then centrifuged at 15000 rpm for 10 minutes. Supernatant was discarded and
pellet resuspended in lysis buffer (appendix), incubated in shaking incubator for 2 hours at
30ᵒC, and followed by centrifugation at 15000 rpm for 20 minutes. Pellet was discarded and
the supernatant containing solubilized toxins which were quantified by method described by
Lowry et al., (1951). SDS-PAGE was used to check the purity of protein toxins.
2.10.2. Protein isolation method 2
A single colony of B.t. from 24 hours old culture was streaked on T3 agar plate and
incubated for 72 hours at 30ᵒC. Streaked plates were then scraped off by using autoclaved
distilled water and centrifuged at 7000 rpm for 15minutes at 4ᵒC. Supernatant was discarded
and pellet was resuspended in autoclaved distilled water and centrifuged at 7000 rpm for 15
minutes. Washing was repeated two times using chilled autoclaved distilled water. Pellet was
resuspended in alkaline buffer (appendix) and incubated in shaking incubator at 37ᵒC for 3
hours. It was then centrifuged at 7000 rpm for 20 minutes at 4ᵒC. Pellet was discarded and
supernatant was saved at -20ᵒC for SDS-PAGE analysis. Protein content of the supernatant
was estimated by Lowry et al., (1951) method.
2.10.3. Sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE)
SDS-PAGE was performed according to method described by Laemmli (1970).
Resolving gel 12% was prepared (appendix) and poured, waited for 15-20 minutes for
complete polymerization at room temperature. Staking gel was prepared (appendix) and
poured on top of the resolving gel and again waited for 15 minutes for polymerization. Before
loading the protein samples were mixed with protein dye (appendix) and heat shock was
given for 10 minutes in boiling water bath. Samples were then loaded and gel run at 40 volts
39
and then at 80 volts. Gel was stained with comassie blue stain and destained with destaining
buffer (appendix).
2.11. Cloning of full length cyt 2B gene
2.11.1. Amplification of full length cyt 2B gene
After confirmation of the presence of cyt 2B gene in the B.t. isolates through
amplification of shorter fragment of the gene, full length gene was amplified.
2.11.1.1. Primer used
For the amplification of full length gene following specific primers were used (Yu et
al., 2002).
Forward primer: GATAATGAGGTTATTTTGTAGAA Tm: 58°C and
Reverse primer: ATCTTACGATTTTATTGGATTAACATTCAGA Tm: 78°C
2.11.1.2. PCR conditions
The reaction mixture includes Taq buffer, MgCl2, dNTPs, Primer forward, Primer
reverse, DNA and ddH2O in a final volume of 50 µl. Amplification was performed in thermal
cycler by using a single initial step at 94ᵒC for 4 minutes, followed by 35 cycles, with each
cycle having denaturation step for 30 seconds at 94ᵒC, annealing for 30 seconds at 52ᵒC and
extension for 1 minute at 72ᵒC followed by a final extension step at 72ᵒC for 10 minutes and
final storage at 4ᵒC (Fig. 2.3). From the PCR mixture, 5 µl were used for agarose gel
electrophoresis.
Denaturation
94 ᵒC 94 ᵒC
Extension
__________ 72 ᵒC 72 ᵒC
5 min 2 min ________
52 ᵒC 2 min 7min
____________
1 min 4 ᵒC
_____
Annealig ∞
Fig. 2.3: Diagrammatic representation of PCR reaction cycle for the
amplification of full length cyt 2B gene.
40
2.11.2. Purification of PCR product of cyt 2B gene
After gel electrophoresis, the full length cyt 2B gene was purified from the gel by
using method described by Sambrook et al., (1998), with some modifications. For
this purpose, Fermentas gene clean kit (#K0153) was used. Under UV light gene band was
cut from the agarose gel and transferred to autoclaved eppendorf tube. To the eppendorf tube
added 3 volumes of NaI and incubated at 55ᵒC for 5 minutes. Removed from incubation and
added 10 µl of silica milk and incubated for 5 minutes at 55ᵒC followed by centrifugation at
10,000 rpm for a time period of 20 seconds. Pellet was saved and supernatant was discarded.
To the pellet added 500 µl wash buffer, 3 washes were given with wash buffer. Air dried the
pellet for 10 minutes and dissolved in autoclaved distilled water (30 µl) followed by
centrifugation at 10,000 rpm for 10 minutes. Supernatant was transferred to a new autoclaved
eppendorf.
2.11.3. Cloning of full length cyt 2B gene
2.11.3.1. Ligation of cyt 2B gene in pTZ57R/T
For this purpose purified PCR product was cloned in pTZ57R/T cloning vector (Fig
2.4) by using Fermentas Ins TAcloneTM
PCR cloning Kit (# K1214). Reaction mixture of 30
µl contained vector (165ng) 3 µl, 5X ligation buffer 6 µl, purified PCR product 4 µl,T4 ligase
(5U) 1 µl and nuclease free water 16 µl. Reaction mixture was then incubated at 16ᵒC for
overnight and then this ligation mixture (pTZ-cyt 2B) was stored at -20ᵒC till further use.
2.11.3.2. Preparation of competent cells
Competent cells of E. coli DH5α were prepared according to method described by
Sambrook and Russel (2001). For this purpose 18 hour old culture was used for inoculating 5
ml autoclaved broth for overnight at 37ᵒC. This overnight culture (500 µl, 1%) was used to
inoculate 50 ml of autoclaved broth and incubated in shaking incubator at 37ᵒC. After 2-3
hours when OD value reached 0.2 to 0.3, the culture was centrifuged at 5400 x g at 4ᵒC for
ten minutes in a sterile falcon tube (50 ml). Supernatant discarded and pellet was resuspended
in ice cold 20 ml CaCl2 (50 mM). This mixture was incubated for 40 minutes on ice and the
centrifuged at 5400x g at 4ᵒC for 10 minutes. Supernatant discarded and pellet resuspended in
4 ml ice cold CaCl2 (50 mM). Cells were streaked on LB agar plate. Till further use cells
were kept on ice.
41
2.11.3.3.Transformation of competent cells (DH5α) with recombinant
plasmid
For this purpose 100, µl of competent cells were mixed with 5 µl of pTZ-cyt 2B
ligation mixture, and kept on ice for 40 minutes. Heat shock was given after ice treatment for
90 sec at 42ᵒC and then cells were shifted quickly to ice for 3 minutes. To this mixture added
800 µl of LB broth and incubated for 1 hour at 37ᵒC followed by centrifugation and resulting
pellet was resuspended in 100 µl of broth. These resuspended cells were spread on LB agar
plates containing Isopropyl-β-D-
thiogalactopyranoside (IPTG) (130µg/ml), X-gal (270µg/ml) and Ampicillin (100µg/ml), and
incubated for overnight at 37ᵒC.
Fig. 2.4: Map of cloning vector pTZ57R/T. The sticky ends with ddT indicated the
cloning sites for the DNA amplicon amplified using Taq DNA polymerase. The vector is
of 2.886 kb with bla as ampicillin resistant marker.
42
2.11.3.4. Selection of transformed recombinant colonies
Selection of recombinant transformed colonies was made on the basis of their
appearance. Colonies that appeared white instead of blue were considered as recombinant
(Sambrook and Russel, 2001). Selected colonies were streaked on LB agar plates containing
100 µg/ml of Ampicillin and incubated overnight. One of the colonies from overnight culture
was used for colony PCR and another for plasmid isolation.
2.11.3.5. Isolation of recombinant plasmid by alkaline lysis method
(Miniprep)
Single colony from pure culture was used to inoculate 5 ml LB broth containing 1
µg/ml of Ampicillin and incubated for 18 hours in shaking incubator at 37ᵒC. Bacterial
culture (approximately 3ml) was transferred to sterile falcon tube and centrifuged.
Supernatant discarded and pellet was suspended in 100 µl of solution I (50 mM glucose, 25
mM Tris-Cl pH 8.0, 10 mM EDTA pH 8.0) and kept for 5 minutes at room temperature and
then shifted on ice. While keeping on ice added 200 µl of solution II (0.2N NaOH, 1% SDS),
mixed gently so that bacterial lysis may
occur. To the lysate added 150 µl of solution III (60% of 5M CH3COOH, 11.5% glacial
acetic acid), vortexed and kept for 3 minutes on ice for protein precipitation. Proteins were
pellet down by centrifugation at 9800 xg for 5 minutes. Pellet discarded and supernatant was
transferred to another eppendorf and equal volume of phenol chloroform (1:1) was added to
the supernatant. This mixture was centrifuged at 9800
xg for 5 minutes. Upper layer was carefully transferred to sterile eppendorf tube and added
twice volume of chilled ethanol. Tubes were kept on ice for 20 minutes followed by
centrifugation at 9800 xg for 10 minutes. Pellets were washed with 70% ice cold ethanol and
air dried in laminar air flow. Pellets were dissolved in 40 µl nuclease free water and stored at
-20ᵒC till further use.
2.11.3.6. Restriction analysis of recombinant plasmid
To confirm the cloning of cyt 2B gene in pTZ57R/T restriction analysis was
performed. Single and double restrictions were performed using EcoR1 and HindIII
restriction enzymes. Reaction mixture for single restriction analysis contained, isolated
plasmid (1.5µg) 5 µl, 10X Tango buffer (Fermentas # BY5) 5µl, 10 U of EcoRI (Fermentas #
ER0271) 1 µl and final volume was raised up to 25µl with nuclease free water. Reaction
mixture for double restriction analysis contained, isolated plasmid 1.5 µg, 10X Tango buffer
(Fermentas # BY5) 5µl, 10 U of EcoRI (Fermentas # ER0271) 1 µl and 10 U of HindIII
(Fermentas # ER0501) 1 µl and final volume was raised up to 25µl with nuclease free water.
Mixtures were then incubated for 3 hours at 37ᵒC. Restricted plasmids were then run on
43
agarose gel (1%) for 45 minutes at 90 volts. To measure the size of restricted bands DNA
ladder mix was used (Fermentas # SM 1173).
2.11.3.7. DNA Sequencing
Cloned genes were sequenced and blasted on NCBI database in order to check their
homology with the already reported genes. Gene sequences were submitted to Gene Bank for
accession numbers.
2.12. Expression of full length cyt 2B gene in E. coli
Full length cyt 2B gene from the six B.t. isolates which were previously cloned in pET
22b were expressed in E. coli for expression. For this purpose E. coli BL21C was used as
host and pET22b was used as expression vectors.
2.12.1. Construction of recombinant DNA
For the cloning of full length cyt 2B gene amplified and purified PCR products were
ligated in pET 22b expression vector (Fig. 2.5) cut with NdeI and BamHI.
2.12.2. Transformation of competent cells (E. coli BL21C)
For this purpose, competent cells (E. coli BL21C) were prepared according to method
describe previously (Sambrook and Russel, 2001). For transformation, 15 µl of recombinant
DNA (pET22b-cyt 2B) were mixed with 200 µl of competent cells in precooled sterile
eppendorf tubes and left on ice for 40 minutes. Cells were quickly shifted to 42ᵒC for 2
minutes for heat shock. Cells were then transferred to ice for 5 minutes. LB medium (1ml)
was added to the tube and incubated for 2 hours without shaking at 37ᵒC. Transformed cells
were spread on LB agar ampicillin (100 µg/ml ) plate and incubated over night at 37ᵒC.
2.12.3. Screening of positive clones
Positive clones were initially selected on the basis of blue white colony. Selected
white colonies were subjected to colony PCR as described previously and restriction
digestion with NdeI and BamHI to confirm 0.8 Kb full length cyt 2B gene.
2.12.4. Expression of recombinant protein
For the expression of recombinant Cyt 2B protein 20 ml pf LB medium supplemented
with ampicillin (100 µg/ml) was inoculated with 1% inoculum
44
Fig. 2.5: Map of expression vector pET 22b.
45
(overnight culture of transformed E. coli BL21C with pET22b-cyt 2B) and incubated at 37ᵒC
till the OD595 reached 0.6. This culture containing transformed E. coli was then induced with
1mM IPTG and incubated for 3 to 7 hours at 37ᵒC in shaking incubator. BL21C transformed
with pET22b without insert was used as control.
2.12.5. Isolation of recombinant protein
The expressed recombinant protein was isolated by taking 1.5ml culture in eppendorf
tube and centrifuged at 13000 rpm for 5 minutes. Supernatant discarded and pellets were
washed with autoclaved distilled water followed by sonication in 140 µl of lysis buffer (1%
SDS, 0.01% β-mercaptoethanol) and then boiled for 10 minutes. During boiling, tubes were
briefly sonicated for solubilization of crystals.
After sonication, tubes were again centrifuged at 13000 rpm. Supernatant was carefully
transferred to another tube without disturbing pellet.
2.12.6. Analysis of expressed recombinant protein
SDS-PAGE was performed for the analysis of expressed recombinant protein. For
this purpose, 15 µl samples and controls were run on 12% SDS-PAGE (12% resolving gel
and 5% stacking gel).
2.12.7. Optimization of conditions for the expression of
recombinant protein
For good expression of recombinant Cyt 2B protein, different conditions like
concentration of IPTG, incubation time and incubation temperature were optimized.
2.12.7.1. Determination of optimum IPTG concentration
For determination of optimum IPTG concentration, different concentrations of IPTG
were used ranging 0.25, 0.5, 1.0, 1.5, 2.5 and 3.0 mM for 3 hours at incubation temperature
of 37ᵒC.
2.12.7.2. Determination of optimum incubation temperature
For determination of optimum incubation temperature, different incubation
temperatures (viz., 28ᵒC, 30ᵒC, 32ᵒC, 35ᵒC, 37ᵒC and 40ᵒC) were tried for 3 hours at IPTG
concentration of 1 mM.
2.12.7.3. Determination of optimum incubation period
For this purpose, different incubation periods viz., 3, 4, 5, 6, 7, 8, 9 and 10 hours were
tried at IPTG concentration of 1 mM and incubation temperature of 37ᵒC.
46
2.13. Purification of expressed Cyt 2B protein
2.13.1. High alkaline pH stress
For the purification of expressed protein high alkaline pH was used because crystal
proteins are soluble at high alkaline pH and other proteins are not. This is why high alkaline
pH was used because it eliminates other unnecessary proteins. For this purpose, high pH
alkaline buffer (30 mM Na2CO3, 20 mM NaHCO3, pH 11.0-11.5) was used. For the
purification of expressed Cyt 2B protein, 1.5 ml culture (OD595 approximately 0.6) with
expressed protein (at IPTG concentration of 1mM and 37ᵒC for 5 hours) was centrifuged at
13000 rpm for 5 minutes. Supernatant discarded and
pellets were washed with autoclaved distilled water and suspended in 200 µl of lysis buffer
by whirl mixing and incubated at shaking incubator at 37ᵒC for 3 hours. After 3 hours, tubes
were again centrifuged at 13000rpm for 10 minutes. Supernatant was carefully transferred to
another tube. Protein concentration was determined by Lowry method (Lowry et al., 1951).
SDS-PAGE was also performed using 15 µl of protein sample.
2.13.2. Anion exchange chromatography
Proteins were purified by method described by Hurley et al., (1987) with some
modifications. After IPTG induction, cultures were solubilized in carbonate buffer (30 mM
Na2CO3, 20 mM NaHCO3, pH 10.0) supplemented with dithiothreitol (10mM) for 3 hours at
37ᵒC. Samples were dialyzed in 10mM phosphate buffer (10mM NaH2PO4
adjusted to pH 7.5 with 1M NaOH) and were then applied to DEAE column (1x30 cm,
Pharmacia) equilibrated with phosphate buffer. After loading of samples bound proteins were
eluted from the anion exchange resin by using KCl gradient (50 mM) in column buffer and
the column flow rate was approximately 15 ml per hour. Protein concentration was
determined by method described by Lowry et al., (1951).
2.14. Biotoxicity assay with expressed Cyt 2B protein
against Ae. aegypti larvae
2.14.1. Bioassay with E. coli transformed with cyt 2b gene
In order to check the toxicity of cloned gene and its expressed protein
bioassays were carried out with transformed organisms against third instar larvae of Ae.
aegypti as described previously. Transformed organisms were first grown in 250 ml LB broth
containing 100 µg/ml Ampicillin and 1mM IPTG and incubated for 7 hours in shaking
incubator at 37ᵒC. Cells were harvested by centrifugation at 10000 rpm and washed two times
47
with autoclaved distilled water. 11 different concentrations were made i.e., 0, 50, 100, 150,
200, 250, 300, 350, 400, 450 and 500 µg/ml.
2.14.2. Bioassay with total expressed proteins of E. coli
transformed with cyt 2b gene
In order to check the toxicity of total expressed protein of E. coli transformed with cyt
2Bgene, bioassays were carried out with total E. coli proteins against third instar larvae of Ae.
aegypti as described previously. Transformed organisms were first grown in 250 ml LB broth
containing 100 µg/ml Ampicillin and 1mM IPTG and incubated for 7 hours in shaking
incubator at 37ᵒC. Total protein was isolated according to method described by Lowry et al.,
(1951). 11 different concentrations were made i.e., 0, 50, 100, 150, 200, 250, 300, 350, 400,
450, 500 µg/ml.
2.14.3. Bioassay with purified expressed Cyt 2B protein
In order to check the toxicity of purified expressed Cyt 2B protein, bioassays were
done with purified Cyt 2B protein against third instar larvae of Ae. aegypti as described
previously. Concentration of purified proteins was estimated by method described by Lowry
et al., (1951). 11 different concentrations were made i.e., 0, 25, 50, 75, 100, 125, 150, 175,
200, 225, 250 µg/ml.
48
RESULTS
3.1. B.t. isolates
A total of 50 soil samples were collected from different areas of Lahore, Kasur and
Faisalabad from different ecological environments under sterile conditions (Table 2.1).
Eleven samples were collected from Kala Shah Kaku agriculture fields and cattle rearing
areas, 10 samples were collected from Pak Arab Society Feroze Pur Road Lahore, 10
samples were collected from Raiwind Road, 2 samples each were collected from Muhammad
Ali Johar Town and canal side near Mall Road Lahore and 5 samples were collected from
country side of Kasur.
Soil samples were incubated in LB broth buffered with 0.2M Sodium acetate. Heat
shock was given for 10 minutes at 80ᵒC to specifically isolate spore formers. LB agar plates
were used for spreading which were then incubated at 30ᵒC for one day. After incubation a
total of 200 B.t. like colonies were appeared and picked from LB agar medium. Out of these
200 initial isolates 73 isolates were identified as B.t. on the basis of different type of staining.
Vegetative cells stained purple with Gram stain and endospores appeared green with
Malachite stain while acid fuchsin stained ICPs with deep pink color.
3.2. Characteristics of B.t. isolates
The 73 endospore former and Gram positive B.t. like isolates were then biochemically
characterized using different biochemical tests (Table 3.1). B.t. isolates were found positive
for catalase activity and were also positive for Voges Proskauer test, can hydrolyze casein
and starch. It can hydrolyze gelatin could decompose tyrosine. It can grow on media
containing 0.001% lysozyme and 7% NaCl. It can also grow on media containing Sabouraud
dextrose. Acid production was observed after glucose utilization. These bacteria could not
grow at 65ᵒC. Some of these bacterial isolates also produced intracellular protein crystals.
Out of 73, 50 B.t. like isolates were selected for further study on the basis of these
biochemical tests (Table 3.2). These 50 out of 73 B.t. like isolates were then subjected to
PCR.
49
Table 3.1. Gram staining, endospore staining and biochemical characterization of local
B.t. like isolates.
Sr.No Parameters Result
1 Gram staining +
2 Endo spore staining +
3 Catalase test +
4 Voges-Proskauer test +
5 Motility test +
6 Casein hydrolysis +
7 Gelatin hydrolysis test +
8 Citrate utilization test +
9 Acid production/ Glucose +
10 Tyrosine decomposition test +
11 Indole test +
12 Growth at Sabouraud Dextrose agar +
13 Nitrate reduction test +
14 Starch hydrolysis test +
15 Growth at 65ᵒC -
16 Growth at 7% NaCl +
17 Growth at .001% lysozyme +
18 Intracellular protein production +
3.3. Prevalence of cyt 2B gene in the B.t. isolates
After the biochemical characterization 50 out of 73 B.t. like isolates were then
subjected to PCR. They were screened for the presence of cyt 2B gene by using already
reported primers. A 469 bp fragment of conserved region of cyt 2B gene was amplified (Fig
3.1). It was found that 37% B.t. isolates were isolated from soil containing cattle waste,
27% were isolated from dry soil, 12% were isolated from
50
Sr. No Texture of soil
sample
B.t. isolates
(%occurrence)
Locality/ Area of collection Positive for cyt 2B
(% occurrence)
1. Saline soil GCU R 1-3 (06%) (i) Road side Qasoor _
2. Sandy soil GCU R 4-9 (12%) (i) Village area Qasoor
(ii) GT road near Faisal abad
_
3. Dry soil GCU R 10-15,25,29,32,36
, 41,42,44,47 (28%)
(i) Pak Arab Ferozep road
(ii) Road side Johar town
(iii) Coriander field near Pak Arab
(iv) Carrot field near Pak Arab
(v) Country side Qasoor
(vi) GT road near KSK
(vii) Rice fields KSK
(viii) Country side Faisal abad
GCU R 11,25,44,47
(27%)
51
1. Moist soil GCU R 19,20,
23,24,37,50 (12%)
(i) Garden soil Johar town
(ii) Canal side near Mal road
(iii) Canal side campus area
(iv) Cattle grazing area Qasoor
(v) Reddish fields near KSK
(vi) Village area Qasoor
GCU R 23,24,37 (20%)
Sr. No Texture of soil
sample
B.t. isolates
(%occurrence)
Locality/ Area of collection Positive for cyt 2B
(% occurrence)
2. Soil containing
Cattle waste
GCU R 16-18,21,22,
26,27,28,30,31,
33-35,38-40,43,
48, 49. (38%)
(i) Ferozepur road near Pak Arab
(ii) Country side Qasoor
(iii) Cattle rearing area Qasoor
(iv) Agriculture field Qasoor
(v) Village area near KSK
(vi) Village area Faisal abad
GCU R
17,18,33,34,38,39,40,49,
(53%)
3. Dry soil with
rice straw
GCU R 39,45,46 (06%) (i) Agriculture field Qasoor
(ii) Rice field KSK
_
52
Fig. 3.1. Agarose gel electrophoresis of PCR product of 469 bp fragment of cyt 2B gene.
Lane 1-5 represent GCU Bt1-5, lane 9 represents GCU Bt 6 and M
represents DNA marker.
moist soil, 12%isolated from sandy soil, 6% were isolated from soil containing rice straw and
another 6% were isolated from saline soil respectively (Fig 3.2 A). PCR
based screening of local B.t. isolates revealed that only 30% isolates were found positive for
cyt 2B gene. Out of these 30% cyt 2B positive isolates, 53% were isolated from soil samples
containing cattle waste, 27% were isolated from dry soil and 20% were isolated from moist
soil, while B.t. isolates isolated from saline soil, sandy soil and soil containing rice straw
were found negative for cyt 2B gene (Fig. 3.2. B).
3.4. Characteristics of cyt 2B positive B.t. isolates
3.4.1. Colony morphology
Colony morphology of cyt 2B positive B.t. isolates were observed. Most of these isolates
were found to have rounded colonies with smooth margins, off white color (Fig 3.3) with rich
growth of colony (Table 3.3).
3.4.2. Microscopic observations
Microscopic observations were done for B.t. isolates positive for cyt 2B gene in order to
check the position of endospore and also to check the presence of para sporal protein
particles. All these isolates were found to be Gram positive purple colored rods. Acid fuchsin
gave deep pink color to protein crystals and Malachite green stained endospores green.
Endospore position was paracentral to sub terminal.
M 1 2 3 4 5 9 8 7 6 10
400bp
53
Fig. 3.2: (A) Frequency of B.t. isolates in different soil samples, (B) Frequency of
cyt 2B positive B.t. isolates in different soil samples.
54
Fig. 3.3. Colony morphology of B.t. isolate positivr for cyt 2B gene.
3.4.2.1. Gram staining
Gram staining of six most toxic B.t. isolates was performed after 18 hours of growth and the
isolates were found to appear as chains of purple colored rods. 3.4.2.2. Endospore staining
Endospore staining of B.t. isolates was performed after 18, 36 and 48 hours of growth.
Vegetative cells were found to sporulate after 18 hours. After 36 hours of incubation, most of
the cells released their endospores into the medium while some vegetative cells still seems to
contain endospores within the vegetative cells. After 48 hours of growth, sporulation seems
to be completed as most of the cells have released their spores into the medium (Fig. 3.4).
3.4.3. Antibiotic resistance of B.t. isolates
It was observed that all the cyt 2B positive isolates were resistant to Ampicillin and
Amoxicillin except GCU R 18,33 and 39 which were found sensitive to Amoxicillin. All
these isolates were sensitive to Erythromycin, Streptomycin, Tetracycline, Vancomycin and
Chloramphenicol except GCU R 18, 33 and 39 which were found resistant to Erythromycin,
GCU R 24, 34, 37, 40 found resistant to Streptomycin, GCU R 37 found resistant to
Tetracycline, GCU R 11 found resistant to Vancomycin (Table 3.4).
55
56
A B
Fig. 3.4. Gram’s staining (A) and endospore (B) staining of GCU B.t. 4.
3.5. Biotoxicity assay with B.t. isolates
A total of 15 B.t. isolates positive for cyt 2B gene (Table 3.2.) were selected to perform
bioassays against 3rd
instar larvae of Ae. aegypti Bioassays were performed by using B.t.
spores and total B.t. cell proteins.
3.5.1. Bioassays with B.t. spores
Bioassays were performed using B.t. spores in order to screen the toxic B.t. isolates
positive for cyt 2B gene against 3rd
instar larvae of Ae. aegypti (Fig. 3.5). GCU B.t. 4 showed
99% mortality at spore dose of 800 µg/ml. At the same spore dose BCU B.t. 1, 2, 3, 5 and 6
showed 94%, 86%, 78%, 90% and 44% mortality, respectively (Table 3.5). Six B.t. isolates
were found less toxic as compared to rest of the B.t. isolates. Among these 6 isolates, GCU
B.t. 7, 10 and 13 showed 100% mortality at spore dose of 1600 µg/ml (Table 3.6) while GCU
B.t. 9, 12 and 15 showed 95%, 96% and 98% mortality at the same spore dose.
Results of spore bioassay indicated that six B.t. isolates were more toxic than rest of
the isolates against 3rd
instar larvae of Ae. aegypti These strains were renamed as NBBt 1
(GCU Bt1), NBBt 2 (GCU Bt2), NBBt 3 (GCU Bt3), NBBt 4 (GCU Bt4), NBBt 5 (GCU
Bt5) and NBBt 6 (GCU Bt6) (Table 3.7).
Table 3.7 also indicates sampling sites from where these samples were collected. LC
50 values of B.t. spores against 3rd
instar larvae of mosquito are shown in Fig 3.5. Bioassay
indicated that GCU B.t.4 is the most toxic B.t. isolate and it was isolated from moist soil rich
in organic manure and has LC 50 value of 400± 1.15 µg/ml. With LC 50 value of 451± 0.90
µg/ml GCU B.t. 1 was isolated from dry soil containing decaying cattle waste with LC50
values of 511± 0.85 µg/ml and 525±.013
57
58
59
A B s
C D
E F
Fig 3.5. Mortality (%) caused by spores of 6 most toxic B.t. isolates GCU Bt 1, GCU
Bt2 , GCU Bt3 , GCU Bt4 , GCU Bt5 and GCU Bt 6, against 3rd
instar larvae of Ae.
aegypti.
60
61
62
µg/ml GCU B.t. 2 and GCU B.t. 5 were isolated from soil containing decaying cattle waste
and from moist soil respectively, with LC50 values of 582± 0.66 µg/ml and 850± 0.34 µg/ml
GCU B.t. 3 and GCU B.t. 6 were isolated from dry and moist soil respectively.
3.5.2. Bioassays with total B.t. cell proteins
The six most toxic B.t. isolates selected after spore bioassay were then used to test the
toxicity of their proteins. For this purpose bioassays were performed using total cell proteins
of these toxic isolates against 3rd
instar larvae of Ae. aegypti Ten different concentrations of
proteins were used ranging between 25 µg/ml – 250 µg/ml for each of the B.t. isolate and as
negative control sodium carbonate buffer was used (Fig. 3.6). GCU B.t.4, with LC 50 value of
68± 0.46 µg/ml was the most toxic B.t. isolate while GCU B.t. 1, showed LC 50 value of 75±
0.95 µg/ml, GCU B.t. 2, 93± 0.88 µg/ml, GCU B.t. 5, 106± 1.32 µg/ml, GCU B.t. 3 106± 0.95
µg/ml and GCU B.t.6, 118± 1.55 µg/ml in decreasing order respectively (Table 3.8).
3.5.3. Comparison of LC 50 of spores and total cell proteins
Toxicity of spores and total B.t. cell proteins of the six most toxic B.t. isolates were
compared (Fig. 3.7). It was observed that total cell protein was found to have more toxic
effect as compared to the spores against 3rd
instar larvae of Ae. aegypti (Table 3.9). Total cell
proteins of GCU B.t.1 showed 6.01 folds increase in toxicity as compared to the toxicity of
its spores. GCU B.t. 2 and 3 showed 5.49 folds while 5.88, 4.95 and 7.20 folds increase in
toxicity of their proteins was observed for GCU B.t. 4, 5 and 6 respectively as compared to
the toxicity of their respective spore doses (Table 3.9).
3.6. Sequencing of shorter fragment of cyt 2B gene
The PCR products of the shorter fragment of cyt 2B gene amplified from the six most
toxic B.t. isolates were them sent for sequencing to ABI sequencers Malaysia. The sequences
of the conserved region of the cyt 2B gene were blast on NCBI database to check their
homology with already reported sequences. These sequences were then submitted to Gene
Bank and were assigned following accession numbers, NBBt1-6, KY777430, KY777431,
KY888138, KY888137, KY888139 and KY777429. Phylogenetic relationship of cyt 2B gene
of B.t.isolate NBBt4 with other
reported cyt 2B gene is indicated in Fig: 3.8 and 3.9.
63
64
A B
C D
E F
Fig. 3.6: Mortality (%) caused by total cell proteins of 6 most toxic B.t. isolates GCU
Bt 1, GCU Bt2 , GCU Bt3 , GCU Bt4 , GCU Bt5 and GCU Bt 6, against 3rd
Instar
Larvae of Ae. Aegypti
65
Fig 3.7. Comparison of LC50 of B.t. spores (Brown) and total B.t. cell proteins
(maroon) against 3rd
instar larvae of Ae. aegypti.
Table 3.9. Comparison of toxicity of spores and total cell protein of B.t. isolates against
3rd
instar larvae of Ae. aegypti.
Sr. No B.t. strain Spores Total cell Increase in
LC50 (µg/ml) Protein Toxicity
LC50 (µg/ml) (Folds)
1 GCU R 11(NB Bt1) 451± 0.90 75± 0.95 6.01
2 GCU R 17(NB Bt2) 511± 0.85 93± 0.88 5.49
3 GCU R 18(NB Bt3) 582± 0.66 106± 0.95 5.49
4 GCU R 23(NB Bt4) 400± 1.15 68± 0.46 5.88
5 GCU R 24(NB Bt5) 525± .013 106± 1.32 4.95
6 GCU R 25(NB Bt6) 850± 0.34 118± 1.55 7.20
0
100
200
300
400
500
600
700
800
900
GCU Bt1 GCU Bt2 GCU Bt3 GCU Bt4 GCU Bt5 GCU Bt6
LC
50
(µ
g/m
l)
Local B.t. isolates
66
Fig 3.8: Phylogenetic relationship of shorter fragment of cyt 2B gene from the most
toxic B.t. isolate NBBt4 with already reported cyt 2B genes.
3.7.1. Nucleotide sequence of shorter fragment of NBBt4 cyt 2B gene
atctttaattgtaatgactaataattgttgttgttgaacatccacagtaatttcaaatgctataggcaatattgccataaatctacct
gtatcttcatttcgaatagaaaataaaattttataaaaataacttatttgagtagatgataagttacgccaaacaatccaattttcat
ctacttgaggttctaaatttgtaaatgtatttgtaatagcagatactacgctgttccaaaaattagcactattgataacgagccctagcacac
ttctgataatttctttaatttgactaatcataactgagacttcaattgtttgatgaattacactatgattaatagttcctgtaa
ctcctgcattaggaagaccatttgcaatttgtaaagctttttcaaaattgaaatttaaggttagtgga
Fig 3.9: Phylogenetic relationship of shorter fragment of cyt 2B gene from the six most
toxic B.t. isolate NBBt1-6 with each other.
3.6. Ribotyping of B.t. isolates
Specific primers were used in order to amplify 16S rRNA gene of the six most toxic
B.t. isolates. The16S rRNA gene is an important tool as it is used to establish relationships
among bacterial phylogeny and is also used to identify unknown bacterium up to genus and
even up to species level. As for as genus Bacillus is concerned it was observed that gene
sequences of 16S rRNA gene of B.t., B. cereus and B. antharacis exhibit high level of
sequence similarity. Amplified PCR products of 1.6Kb of 16S rRNA gene (Fig. 3.10) were
then submitted to Gene Bank after sequencing for the allotment of accession numbers.
Following accession numbers were assigned to B.t. isolates (GCU B.t.1-6) KX611122,
KX611120, KX611121,
KY611803, KY612210 and KY611804 respectively. All these strains showed maximum
homology (99%) with B.t. strain CPB008 16S rRNA gene sequence. Phylogenetic
67
relationship of these B.t. strains has been shown with already reported other strains of B.t.
(Fig 3.11).
Fig: 3.10. Gel electrophoresis of 16S rRNA gene of six most toxic B.t. (Lane 1-6 represents
GCU Bt1-6) isolates M represents DNA marker.
3.6.1. 16S rRNA gene sequence of GCU B.t. 1 (NBBT 1) (KX611122)
AGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGATGGA
TTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAAC
ACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCT
AATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTT
CGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGT
AACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGG
CCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTA
GGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATG
AAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAA
TAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCA
GCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAA
GCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGG
AGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCAT
GTGTAGCGGTGAAATGCGTAGAGAT
TGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACT
GAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGT
AAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCA
TTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACG
GGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCT
TACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCA
1 M 2 3 4 5 6
1.6 Kb
68
GAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGT
CCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTTAGTTGGGCACTCTAAG
GTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCC
CTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACC
GCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTC
GCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGGAATAC
GTTCCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAA
GTCGGTGGGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTGGGA
3.6.2. 16S rRNA gene sequence of GCU B.t. 2(NBBT 2) (KX611120)
CTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAATG
GATTAAGAGCTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGT
GGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATA
CCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGC
TGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACG
GCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCAC
ACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGA
ATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATG
AAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGT
TGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACT
ACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATT
ATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCC
CACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGA
AGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGG
AGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGG
CGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAAC
GATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAA
GCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGG
GGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCT
TACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCTCCTTCGGGAG
CAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT
AAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTTAGTTGGGCAC
TCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCA
TCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAG
CTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGT
AGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCAT
69
GCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGA
GTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTTGGAGCCAGCCGCCTAAGGT
GGGACAGATGATTGGGGTGAAGTCGTAAC
3.6.3. 16S rRNA gene sequence of GCU B.t. 3(NBBT 3) (KX611121)
GGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAA
TGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACA
CGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTA
ATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTC
GGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTA
ACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGC
CACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAG
GGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTG
ATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCT
AGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTA
ACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGA
ATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAA
GCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGC
AGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATA
TGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTG
AGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCA
CGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCT
GAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGA
AACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTT
AATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAG
AGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGC
TCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTT
GCCATCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGT
GGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAAT
GGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTC
TCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCG
CGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAC
ACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTTGGAGCCAGCCG
CCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAAC
70
3.6.4. 16S rRNA gene sequence of GCU B.t. 4(NBBT 4) (KY611803)
CTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAATG
GATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACG
TGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAAT
ACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGG
CTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAAC
GGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCA
CACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGG
AATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAT
GAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAG
TTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAAC
TACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAAT
TATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGC
CCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAG
AAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATG
GAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAG
GCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACG
CCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGA
AGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAA
GGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACG
CGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCTCC
TTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATG
TTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTTAGT
TGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGT
CAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTAC
AAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTC
GGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGA
TCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACC
ACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTTGGAGCCAGCCGC
CTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAA
3.6.5. 16S rRNA gene sequence of GCU B.t. 5 (NBBT 5) (KY612210)
GGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAA
TGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACA
CGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTA
ATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTC
71
GGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTA
ACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGC
CACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAG
GGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTG
ATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCT
AGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTA
ACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGA
ATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAA
GCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGC
AGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATA
TGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTG
AGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCA
CGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCT
GAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGA
AACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTT
AATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAA
CCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATG
GTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGC
GCAACCCTTGATCTTAGTTGCCATCATTTAGTTGGGCACTCTAAGGTGACT
GCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATG
ACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGG
TGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTAC
ATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCC
GGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGT
GGGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGT
CGTAAC
3.6.6. 16S rRNA gene sequence of GCU B.t. 6 (NBBT 6) (KY611804)
CTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAATG
GATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACG
TGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAAT
ACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGG
CTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAAC
GGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCA
CACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGG
AATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAT
GAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAG
72
TTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAAC
TACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAAT
TATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGC
CCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAG
AAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATG
GAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAG
GCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACG
CCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGA
AGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAA
CTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAA
TTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACC
CTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGT
TGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGC
AACCCTTGATCTTAGTTGCCATCATTTAGTTGGGCACTCTAAGGTGACTGC
CGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCT
TATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAA
GACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTA
GGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAG
CATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACAC
CACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTTGGAGCCA
GCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAA
Fig: 3.11. Phylogenetic relationship of 6 most toxic B.t. isolates with already reported
B.t. strains.
3.7. Growth characteristics of selected B.t. isolates
Growth conditions were optimized for the six most toxic B.t. isolates NBBt1-6.
73
3.7.1. Optimum growth temperature For this purpose B.t. isolates were incubated at different temperatures for 24 hours.
Maximum growth of B.t. isolates was observed at 37ᵒC (Fig. 3.12) and was considered as the
best temperature for the growth of B.t. isolates.
3.7.2. Optimum pH for growth
B.t. isolates were grown at different pH conditions. Figure 3.13 depicts the effect of
different pH values on the growth of selected B.t. isolates. It was observed that pH 7 was the
most suited pH for the growth of all B.t. isolates.
3.7.3. Optimum inoculum size
For bacterial growth, quantity of bacterial culture used as inoculum seems to be
critical. Figure 3.14 shows the effect of different concentrations of B.t. as inoculum on the
growth of these isolates. It was found that 8% inoculum size was most suitable for the growth
of all B.t. isolates and no positive effect was observed when inoculum size was increased
from 8% on wards on the growth.
3.7.4. Growth curve of B.t. isolates
To study the growth pattern of selected B.t. isolates Growth curves were plotted.
GCU B.t. 3 showed a very short lag phase and the log phase continued up to 19th
hour
followed by stationary phase. On the other hand GCU B.t. 2 showed typical bacterial growth
pattern. Lag phase lasted for one hour after which bacteria entered into log phase which was
continued up to the 21st
hour leading to stationary phase. GCU B.t.1, 4, 5 and 6 exhibited
almost same growth pattern as that of GCU B.t.2 (Fig. 3.15).
3.8. Molecular characterization of full length cyt 2B gene
For the cloning of full length cyt 2B gene GCU Bt1-6 were selected on the basis of
their biotoxicity, ribotyping and sequences of shorter fragment of cyt 2 B gene.
3.8.1. Amplification of full length cyt 2B gene
PCR conditions were optimized for the amplification of full length cyt 2 B gene.
3.8.2. Cloning and sequencing of full length cyt 2B gene
PCR products of cyt 2 B gene from the six most toxic B.t. isolates shown in Figure
3.16 (A, B) were ligated in PTZ57R (T/A cloning vector). E. coli (DH5α) cells were
transformed with this recombinant DNA. The mixture containing recombinant DNA and E.
coli cells was spread on LB agar plates containing IPTG, X-gal and Ampicillin. Positive
transformants for the presence of cyt 2B gene were screened by
74
A B
C D
E F
Fig 3.12: Effect of temperature on the growth of B.t. isolates
75
A B
C D
E F
Fig 3.13: Effect of pH on the growth of B.t. isolates
76
A B
C D
E F Fig 3.14: Effect of inoculum size on the growth of B.t. isolates
77
A B
C D
E F
Fig 3.15: Growth curves of B.t. isolates A-F (GCU Bt1-6)
78
selecting white colonies containing recombinant plasmid instead of blue. Colony PCR and
restriction digestion using EcoRI and HindIII were done to confirm gene cloning (Fig 3.17,
3.18).
1.0 Kb
Fig. 3.16: (A) Agarose gel electrophoresis of full length cyt 2B gene. Lane 1, 3, 4 and 5
represents GCU Bt1-4 and M represents DNA marker.
Fig. 3.16: (B) Agarose gel electrophoresis of full length cyt 2B gene. Lane 1, 2 represents
GCU Bt 5, 6 and M represents DNA marker.
M 1 5 4 3
M 1 2
1.0 Kb
79
3.8.3. Sequencing of full length cyt 2B gene
After nucleotide sequencing of the 1 Kb full length cyt 2 B gene the sequences of
these isolates were blasted on NCBI database in order to check their homology with already
reported sequences of cyt 2B gene.
Fig. 3.17: Restriction digestion of pTZ57R/T containing cyt 2B gene with EcoRI
and HindIII. Lane 1-6 represents NBBt1-6 and M represents DNA marker.
M 1
Fig. 3.18: Restriction digestion of pET22b containing cyt 2B gene of NBBt4 with
NdeI and BamHI. M represents DNA marker.
1 2 3 4 5 6 M
3.0 Kb
1.0 KB
pET 22b (5Kb)
Cyt 2B gene (0.8 Kb)
80
3.8.4. Expression of full length cyt 2B gene in E.coli (BL21C)
Recombinant DNA consisting of pET 22b expression vector and full length (0.8 Kb) cyt 2B
gene was used to transform E. coli (BL 21C) for the expression of full length cyt 2B gene
through T7 promoter (Fig.3.19).
M
1 2 3 4 5
6
Fig 3.19: Protein profile of B.t. isolates. Lane 1-6 represents NBBt1-6 and M represents
protein marker.
3.8.5. Optimum conditions for the expression of Cyt 2B protein
Conditions of temperature, incubation time and IPTG concentration were optimized
for the good expression of Cyt 2B protein in the host E. coli. It was found that IPTG
concentration of 1mM for a time period of 4 hours was found to be good for the expression of
cyt 2B gene at temperature of 37 ᵒC. For control E. coli transformed with pET 22b without
cyt 2 B gene was used. Total cell proteins were run on 8%SDS-PAGE after isolation. The
expressed 29 kDa full length Cyt 2B protein was observed in protein profile of E. coli
transformed with cyt 2B gene (Fig 3.20). Alignment of the sequence mentioned with already reported sequences in Gene Bank
Database showed maximum homology with AC142305.
30 KDa
81
M 1 2 3 4 5 6 7 8
30 KDa
Fig 3.20: Protein profile of the E. coli transformed with Cyt 2b protein (29 KDa) after
IPTG induction. Lane 2, 4, 6 and 8 after 3, 4, 5, and 6 hours of IPTG induction while
lane 1, 3, 5, and7 represents un induced E. coli after 3, 4, 5, and 6 hours of incubation.
3.8.6. Composition of Cyt 2B proteins of B.t. isolates
The amino acid composition of Cyt 2B protein of six most toxic B.t. isolates was
shown in table 3.10. The amino acid residues of the Cyt 2B proteins were ranged
between 0.4 % of Cys (C) and 11.4% of the Ile (I) out of the total protein content. The amino
acid composition of the 6 most toxic B.t. isolates NBBt1-6 is shown in table 3.10.
3.8.7. Purification of expressed Cyt 2B protein
The over expressed Cyt 2B protein was initially partially purified. For this purpose
protein inclusions were solubilized in carbonate buffer (30 mM Na2CO3, 20 mM NaHCO3,
pH 11.0-11.5) supplemented with DTT (10mM) and incubated at 37 ᵒC for 3 hours. This was
followed by centrifugation at 10000 rpm for 10 minutes at 4 ᵒC. This was done to remove
insoluble proteins because crystal proteins are soluble at high alkaline pH. The crude protein
thus obtained was run on SDS-PAGE to confirm level of purification.
82
83
3.8.8. Anion exchange chromatography of partially purified Cyt 2B
protein
The solubilized partially purified Cyt 2B protein was then subjected to anion
exchange column chromatography. Partially purified protein was dialyzed against phosphate
buffer (10mM NaH2PO4 pH was adjusted to 7.5 by using 1M NaOH). The column flow rate
was 15 ml per hour. Same phosphate buffer was used to equilibrate the DEAE anion in the
column (1x30cm). KCl gradient (1-50mM) was used in the column buffer for the elution of
bound protein and 100 fractions were collected initially. Fractions were then analyzed by
10% SDS-PAGE. It was found that the fractions 45 to 58 contained poly peptide of 29 kDa
representing Cyt 2B protein (Fig 3.21). Protein concentration was determined using lowery
method (Table 3.11). M 1 2
30 KDa
Fig 3.21: Purification of partially purified Cyt 2B protein of NBBt 4,( lane 1 un induced
while lane 2 represents IPTG induction) with anion exchange chromatography. M is
protein marker.
3.8.9. 3D Structure of Cyt 2B protein from NBBt4
Homology based 3D Structure of Cyt 2B protein using deduced amino acid sequence
was constructed. It was found that the structure of Cyt 2Ba and Cyt 2Aa show a similar
topology, consisting of a single α and β domain. α helix are in the form of hairpin loops and
are present in 2 layers wrapping around the β sheet. Among α helix and β sheets only β sheets
are involved in membrane insertion of mosquito gut.
84
Fig 3.22: Homology based 3D Structure of Cyt 2B protein of NBBt4 generated through
EXPACY TOOLS.
Table 3.11. Different steps for purification and yield of Cyt 2B protein of BBt4.
S.No Cyt 2B protein NBBt4
1 O.D of the inoculum of 18 hours 2.5
2 O.D at the time of IPTG induction 0.26
after 3 hours
3 O.D at the time of centrifugation 1.5
after 6 hours
4 Weight of pellet 3.03 g
5 Conc. of crude recombinant 138 mg
Protein
6 Conc. of purified protein 40.4 mg
85
3.8.10. Bioassay with recombinant organism (E. coli BL21C transformed
with plasmid containing cyt 2B gene), expressed crude protein and
expressed and purified protein In order to check the toxicity of individual Cyt2 B protein against Ae. aegypti
bioassay was performed with recombinant organism, expressed crude protein and expressed
purified protein. Different concentrations of recombinant organism, crude protein and
expressed purified protein were made in 20 ml distilled autoclaved water. For each
concentration 20 third instar larvae were used and toxicity was calculated in terms of
percentage mortality. E. coli transformed with plasmid without insert was used as negative
control. It was found that purified expressed protein is most toxic with LC50 value of
50±1.68 µg/ml then the crude recombinant protein with LC50 value of 225±0.66 µg/ml the
recombinant organism was found to be least toxic with LC50 value of 829±0.99 µg/ml
(Table 3.12).
86
87
DISCUSSION
In 1901 Ishiwata Shigetane discovered a disease of Silk worm which was caused by
an un- identified bacteria. E. Berliner named this bacterium as Bacillus thuringiensis in 1911.
Just after 100 years of its discovery 69 % of cotton and 26% of corn planted in USA
contained B.t. genes. Currently various crystal and vegetative insecticidal proteins (cry/vip)
are used to develop transgenic plants resistant to insects. The cry genes isolated till now has
increased to 51 major classes and 349 subclasses (http:/ www.life.sci.sussex. ac.uk/home/Neil
crick more/Bttoxin 2.html).
In the present study, different soil samples were collected for the isolation of B.t.
These include soil from cattle rearing areas, garden soil, dry and moist soil and a large
number of B.t. were isolates from soil rich in organic manure this is in agreement with
Meadows et al., (1992) who reported omnipresent distribution of B.t. The abundance of B.t. in
soil might be due to the presence of enormous amount of nutrients and an increased insect
activity that leads to optimum survival (Al-Momani et al., 2004). B.t. strains have also been
isolated from diverse habitats other than soil, these include aquatic environments (Ishimatsu
et al., 2000), plants (Maduell et al., 2002), insects (Cavadoes et al., 2001), animal faeces (Lee
et al., 2003) and arid environments (Assaeedi et al., 2011). In the Middle East, the natural
occurrence of B.t. in soil environments was reported from Egypt (Merdan and Labib, 2003)
and Jordan (Sadder et al., 2006).
It was found that out of these 50 B.t. isolates 37% were isolated from soil containing
cattle waste, 27% were isolated from dry soil, 12% were isolated from moist soil, 12% were
isolated from sandy soil, 6% were isolated from soil containing rice straw and another 6%
were isolated from soil containing grain dust respectively (Fig 3.2 A). In this study, soil
seems to be a good source of B.t. but it is not in agreement with DeLucca et al., (1981) who
reported only 5% B.t. isolation from soil samples. Theunis et al., (1998) and Apaydin et al.,
(2005), reported grain dust a rich source of B.t. as they found 63% samples of grain dust
positive for B.t. This high percentage of B.t. in grain dust samples might be due to low levels
of humidity and a reduced exposure to UV rays in grain mills (Petras and Casida, 1985;
Ignoffo et al., 1977 Vankova and Purrini, 1979) however in the present study I did not check
the presence of other bacteria in these soil samples containing grain dust because I used
selective media for the isolation of B.t. So I cannot say it with certainty that the absence or
low levels of UV in storage areas has some impact on the occurrence of B.t. their or it may
has an overall effect on the total microbial flora present in the soil samples. In 2005
Balaraman reported the occurrence of B.t. from soils, sediments, animal feces (including wild
mammals, zoo animals and deer), insects (including mosquito larvae and stem borer). In the
present study, 37% B.t. isolates were isolated from the soil rich in organic manure (containing
88
cattle waste). This is in agreement with the findings of who reported the occurrence of B.t. in
four different habitats which are olive fields, grain dust, soil contaminated with industrial
waste products and also from soil contaminated with animal byproducts. In Korea B.t. was
isolated from 18 out of 34 fecal samples of 14 species of wild mammals (Lee et al., 2003).
Prevalence of cyt 2B gene
Now-a-days, PCR is a widely used tool for the characterization of crystal protein
genes and also for the analysis of B.t. isolates for the presence of crystal protein gene. Carozzi
et al., (1991) first time used this technique for the identification of cry genes in order to
predict the insecticidal activity of B.t. isolates. During the last 20 years multiplex PCR
methods are used to identify B.t. isolates harboring cry genes (Ben-Dov et al., 1997, 1999;
Bravo et al., 1998; Ceron et al., 1994; Wang et al., 2003; Ferrandis et al., 1999; Porcar et al.,
2003; Kuo et al., 2000; Uribe et al., 2003). For different subfamilies of cry genes universal
degenerate primers were designed for the amplification of conserved region. As long as the
use of degenerate primers increased the probability of amplifying novel genes but it limits the
detection of closely related genes which are present in the same group (Kuo et al., 2000;
Masson et al., 1998).
In the present study, PCR based screening of local B.t. isolates revealed that only 30%
isolates were found positive for cyt 2B gene. Out of these 30% cyt 2B positive isolates, 53%
were isolated from soil samples containing cattle waste, 27% were isolated from dry soil and
20% were isolated from moist soil, while B.t. isolates isolated from grain dust soil, sandy soil
and soil containing rice straw were found negative for cyt 2B gene. These findings were in
accordance with the findings of Mahalakshmi et al., (2012) who reported the occurrence of
cyt positive strains of B.t. from soil and insects.
Biotoxicity of B.t.
During sporulation B.t. produces proteins which are highly specific in their
insecticidal activity (Hofte and Whiteley, 1989). B.t. is found to have no effect on adult
insects but on the other hand it is very effective against larval or immature stages. In the
toxicity of B.t. δ-endotoxins, its spores play an important contribution (Johnson and
McGaughey, 1996). There are many strains of B.t. which are active against different host
range depending on the type of toxins they have like B.t. var kurstaki is active against
caterpillars, B.t. var israelensis is active against mosquito larvae, larvae of black flies, B.t. var
aizawai is toxic to several species of moths and B.t. var tenebrionis against larvae of leaf
beetles (Schnepf et al., 1998;s deMaagd et al., 2001).
In 2006 use of B.t. var israelensis in mosquitocidal formulations was reported in
standing waters which are the breeding sites of mosquitoes. A new mosquitocidal sub species
of B.t. jegathesan was isolated in Malaysia. Parasporal crystal inclusions of this subspecies
were found toxic against larvae of different mosquito species including Aedes aegypti, Aedes
89
albopictus, Aedes togoi, Anopheles maculatus and Culex quinquefasciatus (Kawalek et al.,
1995). Delecluse et al., (1995) and Orduz et al., (1998) reported that B.t. sub species medellin
is ten times more toxic to mosquito larvae than toxicity of B.t. subspecies israelensis against
mosquito larvae. It was observed that B.t. subspecies kyushuensis and subspecies israelensis
both were found toxic to mosquito larvae (Chilcott et al., 1988; Koni and Ellar, 1994).
In the present study, six most toxic cyt 2B positive isolates of B.t. namely GCU B.t. 4,
1, 2, 5, 3 and 6, respectively showed toxicity against 3rd
instar larvae of mosquito (Ae.
aegypti). Among these isolates GCU B.t. 4 was found to be most toxic as indicated by spore
and protein bioassay with LC50 value of 400± 1.15 and 68± 0.46 respectively. This is in accordance
with the work of Juárez-Pérez et al., (2002), who reported that cyt 2B positive strains of B.t.
are active against mosquito larvae i.e, Ae. aegypti, C. pipiens, C. quinquefasciatus and A.
stephensi.
Wirth et al., (2000) reported the toxicity of cyt 2Ba powders to be more toxic against
C. quinquefasiatus and Ae. aegypti larvae than that of cyt 2Ab. Wirth also reported that cyt
2Ba and cyt 1Ab showed high toxicity when combined with B. sphaericus . cyt 1Ab when
mixed with B. sphaericus in a ratio of 1:3 for a time period of 48 hours this mixture was
found to be most toxic. This mixture suppressed the resistance of mosquito larvae from 17000
folds to only 2 folds against B. sphaericus resistance strain of C. quinquefasiatus. This means
a 7500 fold increase in toxicity against B. sphaericus resistance strain of C. quinquefasiatus.
Even though all these strains used in this study for bioassays were positive for cyt 2B
gene, but they showed different level of toxicity against Ae. aegypti larvae i.e., for the most
toxic strain toxicity of spores was 400± 1.15 µg/ml oUBt4 and 1296± 1.21 µg/ml for
GCUBt12. This difference in toxicity of these isolates may be ascribed to the difference in the
expression level of cyt 2B gene. Likewise the most toxic B.t. isolates in this study were
isolated from soil rich in organic manure and these samples were collected from cattle rearing
areas. These types of habitats are very likely accomplished the rearing of mosquitoes as
compared to other habitats. This indicate that wild animals especially herbivores are naturally
in contact with these bacteria through food intake of plants (Lee et al., 2003).
Mittal et al., (1993) studied the effect of two insecticides Sphaerix (B. sphareicus)
and Bactoculicide (B.t. H14) against larvae of A. stephensi, Ae. aegypti and C.
quinquefasciatus. The toxicity of these insecticides showed variations with the change of
temperature. Sphaerix was found to be almost non- toxic against larvae of A. stephensi and A.
culicifacies at temperature of 21 ± 2ᵒC but at temperature of 31± 2ᵒC against A. stephensi and
A. culicifacies.
Like temperature pH also has effect on the activity of δ-endotoxin. At pH greater than
9, δ-endotoxin produces its subunit called protoxin. It was found that the activity of the δ-
endotoxin is maximum at pH 10-11 (10.5). As for as the structure of these toxins is concerned
it was found that both the protoxin and toxin molecule have α helix and β structure in a ratio
90
of 26% and 45% respectively. At pH 12, it was observed that the toxicity of endotoxin is
decreased and the α-helix content of the toxin both protoxin and toxin also decreased while
the β structure did not change significantly (Venugopal et al., 1992; Feng and Becktel, 1994).
Tran et al., (2001) reported that the insecticidal activity of the protoxin decreased at pH value
lower than 2 and above 11.
This change in toxicity may be attributed to the conformational changes of the toxin
molecules in response to changing pH (Choma and Kaplan., 1990; Convents et al., 1990 Feng
and Becktel, 1994.
Ribotyping of B.t. isolates
16S rRNA gene sequence has been widely used tool for establishing relationship
among different bacteria or we can say that we can establish phylogeny of the desired
bacterium because this 16S rRNA sequence serves as a molecular clock. It is also an
important tool for the identification of unknown bacterium up to genus and even species level.
The B. cereus group includes four species namely B. cereus, B thuringiensis, B.
anthracis and B. mycoides. 16S rRNA sequences of B. cereus, B. thuringiensis, B. anthracis
and B. mycoides exhibit high level of sequence similarity up to 99% with each other
(Henderson et al., 1995). B. cereus is a food pathogen responsible for gastroenteritis
(Drobniewski, 1993) on the other hand B.t. is a bacterium producing bioinsecticide toxic to
many insect larvae (Hofte and Whiteley, 1989).
Traditionally B. anthracis can be distinguished from B. cereus as being non hemolytic
on sheep blood agar, non-motile and consisting of two plasmids coded for virulent toxins and
the presence of capsule. B. mycoides has a unique property in the group as it bears rhizoids
and is motile while B.t. can be separated from B. cereus on the basis of production of
insecticidal crystals inside the vegetative cell during sporulation (Handerson et al., 1995). The
gene for insecticidal crystals is located on plasmid and when any of these strains loses its
plasmid it become very difficult to distinguish it from the B. cereus. The acrystalliferous B.t.
strains which have lost the plasmid bearing genes for crystal proteins it is difficult to
distinguish them from B. cereus (Thorne 1993).
In recent years, molecular methods are major tools in order to specifically identify the
members of B. cereus group. To differentiate B.t. from B. cereus group specific PCR primers
were designed (Yamada et al., 1999). For the analysis of species in the B. cereus group 16S
rRNA sequence was used which indicated that these members are virtually identical with the
variations which are expected for a single species (Saiki et al., 1988; Priest et al., 1994; Giffel
et al., 1997; Yamada et al., 1999).
In the present study, specific primers were used for the amplification of 16S rRNA
gene. 16S rRNA gene sequence is widely used tool now a day to identify a bacterium up to
91
species level. There is a great sequence homology in 16S rRNA gene sequence of some
members of the genus Bacillus namely B.t., B. cereus and B .anthracis, and this homology is
up to 99%. The major difference between B.t. and the other members of the genus Bacillus is
the production of ICPs during sporulation phase (Henderson et al., 1995). After sequence
alignment the 16S rRNA genes of the B.t. isolates were blast on NCBI and their homologies
with other B.t. strains were checked. These sequences were assigned following accession
numbers GCU Bt 1 to GCU Bt 6 as KX611122, KX611120, KX611121, KY611803,
KY612210 and KY611804.
In the present study, it was found that B.t. strains showed maximum homology with
Bacillus thuringiensis serover israelensis strains with accession numbers, AY461762,
EU429672.1, KR109263, KC435169.1, AB617490.1 and JQ669397.1 respectively, already
reported sequences in the Gene Bank database.
Conserved region of cyt 2B gene
The sequences of the conserved region of the cyt 2B gene were blast on NCBI
database to check their homology with already reported sequences. These sequences were
then submitted to Gene bank and were assigned following accession numbers, NBBt1-6,
KY777430, KY777431, KY888138, KY888137, KY888139 and KY777429.
cyt 2B protein
On sporulation, B.t. produces specific larvicidal proteins in the form of parasporal
inclusions (Aronson et al., 1986; Hofte and Whitely, 1989). These inclusion proteins are
made of one or may be several insecticidal proteins also called δ-endotoxins. These δ-
endotoxins are classified into two major classes namely crystal (cry) and cytolytic (cyt)
toxins, on the basis of their amino acid sequence (Crickmore et al., 1998; Hofte and Whiteley,
1989). Most of these δ-endotoxin are synthesized in inactive forms called as pro toxins
present within the inclusion bodies (Aronson et al., 1986).
In this study protein profile of six most toxic B.t. isolates were analyzed by using
SDS-PAGE. In the first method of protein isolation the bands of protein obtained on SDS-
PAGE were of higher molecular weight like 130, 70 and 40 kDa. In the second method high
alkaline pH buffer was used in order to lyse the cells of B.t. The protein bands obtained were
of 130, 68, 40, 29 and 20 kDa. This may be due to the high solubility of the crystal proteins in
the alkaline environment.
Molecular characterization of Cyt 2B protein
Cyt 2B gene is a cytolytic protein gene that is specifically present in B.t. strains that
possess Diptera specific insecticidal activity and especially in the strains which have
mosquitocidal activity. The full length cyt 2B gene is a1 Kb gene which is present on mega
plasmid known as pBtoxis. The full length gene was amplified from all the six most toxic B.t.
isolates namely GCUBt1-6.
92
The nucleotide sequence analysis of the cyt 2B gene using NCBI nucleotide BAST
homology revealed 99% homology with already reported sequence B.t. INTA FJ205865.
Biotoxicity assay
Bioassay were performed with E. coli transformed with cyt 2B gene, with crude
recombinant protein and with expressed purified protein against 3rd
instar larvae of Ae.
aegypti. The LC50 value of transformed organism was 829 µg/ml as compared to the toxicity
of the crude recombinant protein which was 225µg/ml while that of expressed and purified
recombinant protein was 50 µg/ml.
A total of 15 isolates were found positive for cyt 2B gene. These strains were checked
for their mosquitocidal activity and six strains were found more toxic. Among them NBBt4
was found more toxic with LC50 value of 400 ±1.15 µg/ml, 68 ±0.46 µg/ml of its spores and
total cell proteins. Full length cyt 2B gene was amplified from this strain. Cyt 2B full length
gene was then expressed in E.coli BL21C using pET 22b expression vector. Bio toxicity
assay was performed with recombinant organism transformed with cyt 2B gene, expressed
and purified recombinant protein against Ae. aegypti. The purified protein was found to be
most toxic with LC50 value of 50± 1.68 µg/ml. Expressed recombinant Cyt 2B protein can be
used as bio insecticide for mosquito control.
93
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105
Appendix 1
Standard Curve Bradford’s reagent (Bradford, 1976)
S. No
COMPONENTS
Quantity
1 Coomassie blue G250 50mg
2 Methanol 50mL
Dissolve Coomassie blue in methanol to prepare solution1.
3 85% H3PO4 100mL
Added to solution1 resulting in solution2.
4 Distilled water 500mL
Mix well & filter to remove precipitate. Raise volume to 1000mL, store at 4°C.
Procedure Standard assay procedure (for sample with 5-100 µg mL
-1 protein)
C. Prepare five to eight dilutions of a protein (usually BSA) standard with a range of 5 to 100
µg protein. D. Dilute unknown protein samples to obtain 5-100 µg protein/30 µL. E. Add 30 µL each of standard solution or unknown protein sample to an appropriately
labeled test tube.
F. Set two blank tubes. For the standard curve, add 30 µL H2O instead of the standard
solution. For the unknown protein samples, add 30 µL protein preparation buffer instead.
Protein solutions are normally assayed in duplicate or triplicate. G. Add 1.5 mL of Bradford reagent to each tube and mix well. H. Incubate at room temperature (RT) for at least 5 min. I. Absorbance will increase over time; samples should incubate at RT for no more than 1
hour. Measure absorbance at 595 nm.
Standard Curve of Bovine Serum Albumin (BSA)
0.5
0.45
0.4
nm
) 0.35
0.3
(595
0.25
OD
0.2
0.15 0.1
0.05 0 0 20 40 60 80 100 120 140
BSA (µg)
106
Appendix 2
Genomic DNA Extraction and gene amplification
Buffers and chemical preparation used in molecular characterization
1M Tris-HCl Buffer
Chemicals Mass/ Volume
Trizma base 121.14
HCl To adjust pH at 7.6
Sterilized Distilled Water Up to 1000mL
Total 1000mL
0.5M Ethylene Diamine Tetra Acetic Acid (EDTA) Buffer
Chemicals Mass/ Volume
EDTA 186.12
NaOH To homogenize EDTA
Sterilized Distilled Water Up to 1000mL
Total
1000mL
5M NaCl Solution
Chemicals Mass/ Volume
NaCl 292.5
Sterilized Distilled Water Up to 1000mL
Total 1000mL
TEN Buffer (1000 mL)
Chemicals mL
1M Tris-HCl 10
0.5M EDTA 2.0
5M NaCl 2.0
Sterilized Distilled water 1000
SET Buffer (1000 mL)
Chemicals Mass/Volume
1M Tris-HCl 50 mL
0.5M EDTA 100 mL
Sucrose 200 g
Sterilized Distilled water 1000 mL
25% Sodium Dodicyl Sulphate (SDS)
Chemicals
Mass/Volume
SDS 25 g
Sterilized Distilled water 100 mL
107
10X TE Buffer (10 mL)
Chemicals Mass/Volume
Trizma base 0.0012
0.5M EDTA 20 µL
Sterilized Distilled water 10 mL
0.5M Tris-HCl Buffer
Chemicals Mass/ Volume
Trizma base 60.05
HCl To adjust pH at 7.6
Sterilized Distilled Water Up to 1000mL
Total 1000mL
0.1M Tris-HCl
Chemicals Mass/ Volume
Trizma base 12.114
HCl To adjust pH at 7.6
Sterilized Distilled Water Up to 1000mL
Total 1000mL
TAE Buffer 50X
Chemical Concentration Mass/ Volume
Trizma base 2M 242g
EDTA 0.5M 37.2g
Glacial Acetic Acid 5.71% 57.1mL
Total volume 1000mL
1X TAE Buffer
20 mL of 50X TAE buffer was diluted to 1000 mL with distilled water to prepare 1X Tank buffer.
Chloroform:Isoamyl alcohol (24:1)
Chemicals Mass/ Volume
Chloroform 25mL
Ethanol 1mL
Freshly prepared prior to use.
70% Ethanol
Chemicals
Mass/ Volume
Ethanol 7.0mL
Distilled water 3.0mL
Freshly prepared prior to use.
108
Agarose Gel Composition
Tray
1X TAE Buffer
1% Agarose
1.5% Agarose
7×10cm 50mL 0.5g 0.75g
10×10cm 75mL 0.75g 11.25g
15×10cm 100mL 1g 1.5g
DNA Loading Dye
Chemical
Concentration
Mass/ Volume
Glycerol 50% 5mL
CTAB Buffer 1X 200µL of 50X Stock
Bromophenol Blue 1% 0.1g
Xylene Cyanol 1% 0.1%
Total volume 10mL
Ethidium Bromide Solution
Chemical
Mass/ Volume
Ethidium bromide 0.01g
Glycerol 1mL
3µL of Ethidium bromide solution was added in an agarose gel of 50mL.
Amplification PCR Reaction Mixture
Chemicals
Concentration
Mass/ Volume
Taq Buffer 1X 1.25 µL
MgCl2 2 mM 0.5 µL
dNTPs 0.12mM 0.3 µL
Primer-forward 0.2µM 0.25 µL
Primer-reverse 0.2µM 0.25 µL
BSA 8 µg/mL 0.1 µL
dd H2O 7.55 µL
Total volume 12.5 µL
109
DNA Ladder
A DNA Ladder of 1 kb plus 100 bp was used for comparison of DNA bands. Cat. Number for this marker was
―SM-0313‖
Extraction of gDNA of bacterial strains
For the extraction of bacterial genomic DNA, phenol-chloroform method was used.
Steps involved in gDNA extraction were as follows:
1. Lauria-Burtani broth medium was inoculated with an isolated colony of pre-cultured bacteria and was incubated
at 37°C overnight under shaking conditions.
2. Cellular pellet was obtained by centrifugation of 10 mL of culture medium at 10,000 rpm for 20 minutes.
3. Supernatant was discarded and the pellet was suspended in 400µL of TEN buffer. Pellet was centrifuged at
10,000 rpm for 10 minutes.
4. Supernatant was discarded and the pellet was resuspended in SET buffer (200µL), followed by 120µL of
Lysozyme (20mg/mL).
5. The reaction mixture was incubated at 37°C for half an hour.
6. Afterwards, 200µL of TEN buffer and 10µL of 25% SDS solution was added and incubated.
7. After incubation at 60°C for 15 minutes, the mixture was allowed to cool to room temperature. Twenty µL of
5M NaCl solution was followed by an equal volume of phenol:chloroform mixture (1:1)
8. It was centrifuged at 10,000 rpm for 20 minutes leading to a prominent aqueous layer above the matt of
degraded proteins.
9. The aqueous layer was transferred to a new pre-labeled tube containing an equal volume of chloroform.
10. Centrifugation at 10,000 rpm for 15 minutes resulted in separation of aqueous layer from chloroform.
11. Again, the top layer was transferred to new tubes and incubated overnight with double volume of isopropanol.
12. Afterwards, tubes were centrifuged again at 10,000 rpm for 10 minutes and supernatant was discarded.
13. Pellet was washed twice with 70% ethanol and air dried.
14. DNA pellet was stored at -20°C in 50µL of deionized water or TE buffer until further use.
110
Appendix 3
Polyacrylamide Native Gel (Laemmli, 1970)
I- Reagents
Acrylamide-Bisacrylamide stock solution
Chemicals Mass/ Volume
Acrylamide 30
Bis-acrylamide 0.8
Distilled water (raise volume) 100mL
After raising volume with distilled deionized water, it was stirred for 4 hours.
Stacking gel buffer_0.5M Tris-HCl (500mL)
Chemicals Mass/ Volume
Tris base 30.29g
Distilled water 500 mL
Tris base was dissolved in 300mL distilled water and pH was adjusted to 6.8 while using 1M HCl. Volume raised to 500mL.
Resolving gel buffer_3M Tris-HCl (500mL)
Chemicals Mass/ Volume
Tris base 181.71g
Distilled water 500 mL
Tris base was dissolved in 300mL distilled water and pH was adjusted to 8.8 while using 1M HCl. Volume raised to 500mL.
5X Reservoir buffer Native-PAGE (pH 8.3)
Chemicals
Mass/ Volume
Tris base 3.03g
Glycine 14.4 g
ddH2O Up to 1000 mL
5X Reservoir buffer SDS-PAGE (pH 8.3)
Chemicals
Mass/ Volume
Tris base 3.03g
Glycine 14.4 g
SDS 1.0 g
ddH2O Up to 1000 mL
Freshly prepared 10% Ammonium per sulphate (APS)
TEMED (N, N, N: N’ – tetramethyl ethylene diamine)_used as such
0.5% Bromophenol Blue
10% SDS
Glycerol _used as such Β-mercaptoethanol_used as such
111
0.04% Coomassie brilliant blue G-250
Chemicals
Mass/ Volume
Coomassie brilliant blue G-250 40mg
3.5% perchloric acid 100mL
Solution was stirred for an hour on a magnetic stirrer and filtered through Whatman No. 1 filter paper and stored in amber colored bottle at room temperature.
Destain solution_7.5% Acetic acid
II- Native PAGE
Stacking gel composition
Chemicals Volume
Acrylamide- Bis-acrylamide 2.5mL
Stacking gel buffer 5.0mL
Distilled water 11.5mL
APS 0.1mL
TEMED 0.02mL
Resolving gel composition_12.5%
Chemicals Volume
Acrylamide- Bis-acrylamide 12.5mL
Resolving gel buffer 3.75mL
Distilled water 14.25mL
APS 0.15mL
TEMED 0.02mL
III- SDS-PAGE
Stacking gel composition
Chemicals Volume
Acrylamide- Bis-acrylamide 2.5mL
Stacking gel buffer 5.0mL
SDS 0.2mL
Distilled water 11.3mL
APS 0.1mL
TEMED 0.02mL
112
Resolving gel composition_12.5%
Chemicals Volume
Acrylamide- Bis-acrylamide 12.5mL
Resolving gel buffer 3.75mL
SDS 0.3mL
Distilled water 12.0mL
APS 0.15mL
TEMED 0.02mL
IV- Sample preparation
Sample buffer_Native PAGE
Chemicals Volume
Distilled water 3.0mL
Stacking gel buffer 1.0mL
Bromophenol blue 0.4mL
Glycerol 1.6mL
Sample buffer_SDS-PAGE
Chemicals Volume
Distilled water 3.0mL
Stacking gel buffer 1.0mL
Bromophenol blue 0.4mL
Glycerol 1.6mL
SDS 1.6mL
Β-mercaptoethanol 0.4mL
Procedure
1. For Native PAGE sample and sample buffer taken in 1:1 proportion. 2. For SDS PAGE sample and sample buffer taken in 1:1 proportion and heated in a
boiling water bath for 3 minutes.
Protein molecular weight marker A Protein Ladder of 100 kDa plus was used for comparison of protein bands. Cat. Number for this marker was
―SM-0671/2‖
113
Electrophoresis conditions
During electrophoresis, a constant voltage of 80V was given at the time of
pre-running. After the sample has been loaded the voltage was maintained at 70V.