APCBEE Procedia 2 ( 2012 ) 165 – 169

2212-6708 © 2012 Published by Elsevier B.V. Selection and/or peer review under responsibility of Asia-Pacific Chemical, Biological & Environmental Engineering Societydoi: 10.1016/j.apcbee.2012.06.030

ICBFS 2012: April 7-8, 2012, Bangkok, Thailand

Isolation and Identification of Helicase from Escherichia coli for Biotechnology Processes

S.Sasidharan∗, S.Yamuna, O. Eugene, B.Venugopal, I.Asma Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 USM, Pulau Pinang ,Malaysia

Abstract

Helicases are able to unwind duplex DNA enzymatically at one temperature without prior heat denaturation. Therefore, the current study was undertaken to isolate and identify E. coli for in house production of helicase enzymes. Bacteria were isolated from patient stool samples from Penang General Hospital, Penang, Malaysia. The light and scanning microscopy technique were used to identify the morphology of the isolate. Chromosomal DNA from the organism was isolated and used to amplify 16S rRNA and uvrD gene fragments. The gene was amplified by a set of universal primers (F_UNI16S and R_UNI16S). The phylogenetic tree, homological analysis, and detailed comparison of the sequences showed that 16S rRNA gene sequence of the isolate had closest similarities with E. coli. Isolates gave PCR fragments of 2163bp which represent the uvrD gene and 1500bp which represent the 16S rRNA respectively of E. coli. E. coli was successfully isolated and identified by using 16S rRNA gene sequence analysis with uvrD gene © 2012 Published by Elsevier B.V. Selection and/or peer review under responsibility of Asia-Pacific Chemical, Biological & Environmental Engineering Society Keywords: Helicase, local isolates and UrvD gene.

1. Introduction

Polymerase chain reaction (PCR) is the extensively employed method for in vitro DNA amplification (Belanger et al., 2003). However, it requires thermocycling to separate two DNA strands and well-trained

personnel for operation. It has a significant benefit over PCR in that it get rid of the need for an expensive and power-hungry thermocycler. DNA helicases catalyze the conversion of double stranded DNAs into single

*Corresponding author: Tel :+6046534820 ; Fax: 6046534803 E-mail address: srisasidharan@yahoo.com

Available online at www.sciencedirect.com

© 2012 Published by Elsevier B.V. Selection and/or peer review under responsibility of Asia-Pacifi cChemical, Biological & Environmental Engineering Society Open access under CC BY-NC-ND license.

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166 S. Sasidharan et al. / APCBEE Procedia 2 ( 2012 ) 165 – 169

stranded forms, making the bases accessible to various DNA metabolic processes, i.e., replication, recombination, and repair. Helicase-dependent amplification (HDA) is a unique isothermal nucleic acid amplification technique that relies on the use of a DNA helicase enzyme to unwind double-stranded DNA and RNA-DNA hybrid. HDA is an isothermal in vitro DNA amplification method based upon the coordinated actions of helicases to separate double-stranded DNA and DNA polymerases to synthesize DNA. Helicases also function in other cellular processes where double-stranded DNA must be separated, including DNA repair and transcription. Nucleic acid amplification through HDA is very robust, and the platform can be applied to both DNA and RNA pathogen amplification (An et al., 2005; Goldmeyer et al., 2007). The current study was proposed for isolation identification local clinical isolate of E. coli from patient with fever to obtain a strain producing a temperature-labile helicase which is able to grow at elevated temperature.

2. Experimental Procedure

2.1. Sampling of microorganisms

200 l watery stool material was inoculated into 5 ml trypticase soy broth and incubated at 37 °C overnight. This enrichment culture was plated onto Sorbitol-MacConkey Agar (SMAC) and MacConkey Agar (MAC) plates. Ten random lactose-fermenting colonies were picked from the MAC plates and used for ribotyping identification of a helicase producer E. coli.

2.2. Characterization and identification of the isolates

2.2.1. Morphological Studies Morphological properties were investigated by using 18 hour old bacterial cultures growing on nutrient

agar plates. These included the wet mount preparations using light microscope Gram staining to confirm Gram reaction. Light micrograph and scanning electron microscopy were performed to identify the ultrastructure of the bacteria.

2.3. Identification of Helicase.

UvrD gene sequence analysis was used in order to detect the presence of uvrD Helicase gene and 16srRNA (ribotyping method) to identify E. coli. The reaction mixture was set up on ice as follows: 5.0 l 10x PCR Buffer contains Tris.Cl,KCl, (NH4)2SO4,15mM MgCl2, stabilizers with ph of 8.7 (20°C), 10 l 5x Q-solution ,19.75

l nucleus free water , 1.0 l dNTP(10mM), 1.0 l Primer F_UNI16S (10pmol/ l), 1.0 l Primer R_UNI16S (10pmol/ l), 1.0 l Primer E_coli_uvrD_F(10pmol/ l) and 1.0 l Primer E_coli_uvrD_R(10pmol/ l),, 0.25 l Taq polymerase and 1.0 l DNA template(50ng/ l) with 50 l final volume. The PCR program was initial denaturation 3 minutes at 94°C, 35 cycles: 30 sec at 94°C; 30 sec at 60°C; 1 minute at 72°C; followed by a single period at 72°C for 10 minutes.

3. Results and Discussion

E. coli were isolated from Penang General Hospital. Isolation was performed by sampling Gram- lactose-fermenting pink typical coliform colonies MAC plates. Morphological observation of E. coli under light and scanning microscopy showed that the cell is a rod shaped bacteria (Figure 1a & b). Further analysis using gram-staining method proved that the isolate was a gram-negative bacterium (Figure 1a). Table 1 shows the primer and PCR condition used for the detection of 16S rRNA gene of isolates and UvrD gene. 16S rRNA

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gene amplified with F_UNI16S and R_UNI16S primers from E. coli isolate was observed on agarose gel electrophoresis. The result showed two amplicon bands with the size of 2163bp which represent the uvrD gene and 1500bp which represent the 16S rRNA consequently of E. coli respectively (Figure 2 to Figure 4).

Table 1. Primers and PCR conditions used for detection of uvrD gene

Primers Used PCR conditions No. of cycles

UvrD E_coli_uvrD_F 5’- GAC GAG CTC GAC GTT TCT TAC CTGCT – 3’ Sac1

E_coli_uvrD_R 5’- TAT CTC GAG TTA CAC CGA CTC CAG CCG- 3’ Xho1

Initial Denaturation 94οC – 3min

Denaturation 94οC – 30 sec

Annealing 60οC – 30sec

Extension 72οC – 1min Final Extension 72οC – 10 min

Hold 5οC –

16SrRNA F_UNI16S 5’- AGA GTT TGA TCC TGG CTC AG – 3’

F_UNI16S 5’- AAG GAG GTG ATC CAG CC -3’

Initial Denaturation 94οC – 3min

Denaturation 94οC – 30 sec

Annealing 60οC – 30sec

Extension 72οC – 1min Final Extension 72οC – 10 min

Hold 5οC –

PCR products were visualized by electrophoresis using 1% agarose gel. E.coli strains were subjected to

PCR for helicase gene and 16SrRNA gene identification.

Fig. 1. (a) Morphology of the E. coli under the Light Microscope with 100X amplification.; (b) SEM micrograph of E.coli sp.

35

35

a b

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Fig. 2. Amplification of 16S rRNA .Isolates gave PCR fragments of 1.5kb which represent the entire 16S rRNA gene. Lanes: M, 1kb DNA ladder; 1 until 8 ,the E.coli Isolates with different template concentrations.

Fig. 3. Amplification of uvrD gene .Isolates gave PCR fragments of 2163bp which represent the uvrD gene of E.coli sp.. Lanes: M, 1kb DNA ladder; 1 until 8 ,the E.coli Isolates with different template concentration.

Fig. 4. Amplification of uvrD gene and 16S rRNA gene from E.coli sp .Isolates gave PCR fragments of 2163bp which represent the uvrD gene and 1500bp which represent the 16S rRNA consequently of E.coli sp. using multiplex PCR. Lanes: M, 1kb DNA ladder; 1 until 8 ,the E.coli Isolates with different template concentration.

The polymerase chain reaction (PCR) modernized the abilities to performs biotechnological research, and it has been widely used in biomedical research and disease diagnostics (Saiki et al, 1988). Hand-held diagnostic devices, which can be used to detect pathogens in the field and at point-of-care, are demanded currently especially in rural area of third world countries. However, the need for power-hungry thermocycling limits PCR application in such a situation. Helicase-dependent amplification (HDA) devised a new isothermal DNA amplification technology could solved this short coming. As the DNA helicase unwinds dsDNA

1500bp

2163bp

2163 bp

1500 bp

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enzymatically, the initial heat denaturation and subsequent thermocycling steps required by PCR can all be omitted. In this study, we isolated the Escherichia coli with UvrD-based HDA system, which can produce helicase in house. In this study, we report a new local Isolates with Helicase. It has a significant advantage over PCR in that it eliminates the need for an expensive and power-hungry thermocycler. As DNA helicase can melt double-stranded target DNA at the beginning of the reaction, the entire HDA reaction can be performed at one temperature (Vincent et al, 2004).

4. Conclusion

In Conclusion, based on the results of this study, the isolated strain showed presence of E.coli with UvrD gene and was successfully isolated from patient with high fever that showed Gram–negative rods and aerobic characteristics. The straightforwardness and true isothermal nature of the HDA platform tender great potential for the development of hand-held DNA diagnostic devices that could be used to detect human pathogens at point-of-care or in the field especially for the poor people in the third world country.

Acknowledgements

This project was funded by USM Research Grant RUC (1001/PSKBP/86300110) from Universiti Sains Malaysia.

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