a u s t r a l i a ’ s g r o w i n g f u t u r e the xylanase transient quantification system...
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A u s t r a l i a ’ s G r o w i n g F u t u r e
The Xylanase Transient Quantification System
Claudia Vickers
Transient vs. Stable Transformation
• Transient transformation– DNA extra-chromosomal– Seen over several days following transformation
• Stable transformation– DNA integrated into chromosome– Demonstrated by transferral of transgene to offspring– Segregation occurs in offspring
Transient Transformation as a Tool
• Quick and easy (days compared to months)
• Not prone to position-of-integration effects
• Cheap and less labour-intensive than producing transgenics (esp. cereals)
• Measure tissue specificity and expression strength
• But – expression patterns not always reflected in stable transgenics
Reporter Genes
• Genes which allow easy detection of expression to confirm occurrence of transformation
• Different reporters useful for different experiments
• Properties of the reporter gene must be tuned to the experiment
Desirable Features
• Short coding sequence (easy to manipulate)
• Low (preferably no) endogenous activity in plants
• No endogenous substrates in plants
• Easy, quantitative assay
• Cell autonomous
• Tolerate terminal fusions (purification)
• Active and stable under a range of cellular conditions
• Cheap!!!
Reporter Genes
GENE PRODUCT DETECTION
PROS CONS
GUS(uidA)
-glucuronidase
Colour reaction, fluorescence
Extremely well characterisedExcellent assay systems availableQualitative, quantitative
Many reports of endogenous GUS-like activityProblems with cell autonomyQuenching
LUC(luc)
Luciferase(firefly/ bacterial)
Luminescence (light emission)
Extremely sensitiveQualitative, quantitative
Very expensiveVery labileCannot increase sensitivity
antRC
Regulatory proteins
Anthocyanin pigment
Cell-autonomousNo substrate requiredNon-destructive
Not for quantificationToxicity problems
GFP(gfp)
Green fluorescent protein
Fluorescence
Cell-autonomousNon-destructiveNo substrate requiredPictures look great!
Problems with quantificationChlorophyll autofluorescence
Transient Analysis
• Tissue specificity– How many tissues??– Limited to tissues that can be transformed– Tissue specificity may require chromosomal integration– Most reporters will suffice
• Quantification– Assess promoter strength– Test effect of introns, enhancers, 5 and 3 UTRs etc.– Not all reporters are suitable
Expression Strength
1. Focus counting– Co-transform with GFP and GUS plasmids– Count foci (GFP and GUS)– Expression strength = ratio of GFP:GUS– Problems with threshold effects
Focus Counting: Threshold Effect
0 bp3009001200 bp 600 0 0.5 1 1.5GFP:GUS
Barley
Wheat
GFPXE BMS
TSS
UTR
D1 D3
Actin
OG
OG D1
OG D3
OG X
OG S
OG M
Barley
Wheat
Focus Counting: Threshold Effect
0
10
20
30
40
50
60
70
80
P1 P2 P3 P4 P5 P6 P7
Fo
cus
Inte
nsi
ty (
RL
U)
Detection threshold
Saturation threshold
Focus Counting – Threshold Effect
1 2 3 4 5 6
Promoter
1 2 3 4 5 6
Promoter
Fo
cus
Inte
nsi
ty
0
20
40
60
80
Nu
mb
er o
f F
oci
Average Focus Insensity
Number of Foci
B
Expression Strength
• Focus counting– Co-transform with GFP and GUS plasmids– Count foci (GFP and GUS)– Expression strength = ratio of GFP:GUS– Problems with threshold effects
• Protein extraction and reporter quantification– Shoot with GUS and LUC plasmids– Extract protein from tissues– Quantitative reporter gene assays– Expression strength = ratio of reporter activity– Problems….
Schledzewski & Mendel 1994
Reporters for Quantitative Assay
• GUS: Fluorometric, time-response assay– Very sensitive (can increase incubation time)– Slope over time – good statistical significance– Problems with endogenous activity and/or quenching
(particularly in leaf extracts)
• LUC: Luminometric assay– Extremely labile– No option to increase sensitivity– Expensive (substrate, co-factors, detection equipment)
Solution:
• Thermostable XYN developed by gene shuffling• Codon optimised• Stable transformants generated• Time-response assays possible (increase sensitivity)• Substrate:AZCL-xylan
– Minor problem: insoluble– Other potential substrates: 4-MU-xylan (fluorometric), soluble
colorimetric substitute
• Cheap Cheap Cheap (like the birdie)!• Used in concert with GUSPlus greatly incr. sensitivity
XylanaseXylanase
Physical Optima
• pH = 4.5
• Temp. = 40C
• Linear response to enzyme concentration
• Substrate saturates at 0.5%
pH Response @ 40 C
0
1
2
3
4
3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
pH
Ab
sorb
ance
(59
0 n
m)
Temperature Response @ pH = 4.5
0
1
2
3
4
0 20 40 60 80
Temperature (Degrees C)
Ab
so
rba
nc
e @
59
0 n
m
Enzyme Concentration
R2 = 0.9924
0
1
2
3
4
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1
Extract Dilution
Ab
sorb
ance
@ 5
90 n
m
Substrate Concentration
0.0
0.5
1.0
1.5
2.0
2.5
0 0.5 1 1.5 2 2.5
AZCL-Xylan (%)
Ab
sorb
ance
@ 5
90 n
m
Time Response
Time Response - P/100
y = 0.0004x + 0.0377
R2 = 0.99090.00
0.20
0.40
0.60
0.80
0 500 1000 1500 2000
Time (min)
Ab
so
rba
nc
e (
A5
70
)
• Linear over time
• Up to 48 hr
• Allows accurate quantification of weak promoters
High-Throughput
• Colour intensity for each well depends on:– Transformation efficiency
– Concentration of sample
– Time of incubation
• Standardised to internal control (GUS)
Deletion Analysis- Focus Counting
Actin
OG
OG X
OG S
OG M
OG D3
OG D1
0 bp3009001200 bp 600 0 0.5 1 1.5GFP:GUS
Barley
Wheat
GFPXE BMS
TSS
UTR
D1 D3
Actin
OG
OG D1
OG D3
OG X
OG S
OG M
Barley
Wheat
Deletion Analysis – GUS:XYN
100 200 300 400 500
GUSPlus :Xylanase100 200 300 400 500
GUSPlus :Xylanase
0 bp3009001200 bp 600 0 bp3009001200 bp 600 0 bp3009001200 bp 600
Globulin
Hordein
Glo X
Glo S
Glo M
Negative
Glo D1
Glo D2
Glo D3
Summary: XYN/GUSPlus
• Quick (transient vs. stable)• User-friendly• Accurate• Cheap• Sensitive
Vickers, C.E.; Xue, G.-P.; Gresshoff, P.M. (2003) A synthetic xylanase as a novel reporter in plants. Plant Cell Rep. 22(2):135-140
Xylanase Assaysin Plant TissuesClaudia Vickers [[email protected]]
Caveat: It is assumed that the readers have read the paper entitled, ”A synthetic xylanase as a novel reporter in plants” (Plant Cell Reports 22(2):135-140, 2003) before reading these notes.
IntroductionReporter Genes UsedDetailed MethodSolutionsReferences
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IntroductionQuantification of the effect of promoter regions on reporter gene expression in transient assays can be used to gain information about how a particular section of DNA drives gene expression in isolation from a chromosomal context. There are advantages and disadvantages associated with using transient analysis. The obvious disadvantage is that promoters do not always behave in the same fashion when integrated into plant DNA. Conversely, one can obtain data that is not affected by position-of-integration effects. In addition, the method is quick and simple compared to the production of transgenic plants. As noted previously, this is particularly useful when investigating promoter activity in species for which the transformation efficiency is relatively low.
Accurate quantification of transient expression requires extraction of protein and quantitative analysis of reporter gene activity. Extremely high sensitivity of reporter gene assays is required because of the dilution of the gene product during protein extraction (the vast majority of cells in bombarded tissues are untransformed). This is generally achieved through extreme sensitivity of detection of the reporter gene product or of the product of the reporter gene’s activity. Enzymatic activity is a practical necessity for this kind of detection. In practice, this results in amplification of the signal. Enzymatic systems that allow extension of incubation periods and consequent accumulation of the cleavage product are preferable, as this allows for further increases in assay sensitivity.
http://www.pi.csiro.au/XylanaseAssays/index.htm
Ordering the XYN Assay Kit
PLASMID ORDER FORM
A charge of AUD $100 per package is made solely to cover costs of production and postage. Please return this form, together with the attached materials transfer agreement, to the ARC Centre of Excellence for Integrative Legume Research by fax or post. Please print clearly. Name: Dr/ Mr/ Mrs / Ms _____________________________________________________________________
Institute: ______________________________________________________________________________
Postal Address: ______________________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
Street Address: ______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
Phone: __________________________________
Fax: __________________________________
Email: __________________________________
Plasmids (pUbiSXR, pActXYN and pUbiGUSPlus) are supplied as 100 ng samples blotted on paper. Please note that we cannot send plasmids until a signed copy of the attached materials transfer agreement has been received. An invoice for AUD $100 will be included with the package. Please return signed and dated form to Ian Harris, Chief Operating Officer, Centre for Integrative Legume Research, John Hines Building (62), The University of Queensland, Brisbane QLD AUSTRALIA 4072. Forms may be faxed to +61 7 3365 3556.
ARC Centre of Excellence for Integrative Legume Research
John Hines Building (62) The University of Queensland St Lucia Brisbane, AUSTRALIA 4072 Ph: +61 7 3365 3550 Fax: +61 7 3365 3556 Email: [email protected]
MATERIAL TRANSFER AGREEMENT (MTA)
This material transfer agreement (MTA) is made between (name)_____________________________________,
an employee of (institute and street address) ____________________________________________________
__________________________________________________________________________________________
(‘the Recipient’) and The Centre for Integrative Legume Research at the University of Queensland (“the University”). This MTA covers transfer of the following Materials:
Xylanase assay plasmid vector kit:
- pUbiSXR - pActXYN - pUbiGUSPlus
This material is provided with no warranties of any kind, express or implied, and with no representation that use or supply of these materials will not infringe the rights of any third party, including intellectual property ri ghts. To the full extent permitted by the laws of Australia or of any state or territory of Australia having jurisdiction, any conditions or warranties imposed under legislation are hereby excluded. The University and its employees, past or present, cannot be held liable for any use of the material transferred according to this agreement. The Recipient agrees to the following conditions:
1. These plasmid vectors may be used freely for academic and non-profit research. 2. Use shall be duly acknowledged citing this internal non-commercial non-exclusive license from the
University. 3. Commercial Use: A license from the University is required (i) for any commercial use of the plasmid
vectors including research and production purposes regardless of academic or non-profit status and (ii) for use by any commercial entity. Information about commercial licenses for the Plasmid Vectors may be obtained from CILR at The University of Queensland
4. Plasmids may be distributed to third parties, however, the University must be notified prior to transfer and a copy of this MTA must be signed and returned to the University before the plasmids are transferred.
5. Use of the xylanase (XYN) gene must be acknowledged in resulting publications. The citation is: Vickers, C.E.; Xue, G.P.; Gresshoff, P.M. (2003) A synthetic xylanase as a novel reporter in plants. Plant Cell Reports 22[2]: 135-140
6. The GUSPlus gene in pUbiGUSPlus is protected by patents belonging to CAMBIA. Terms for the use of this gene can be obtained from CAMBIA (GPO Box 3200, Canberra, ACT 2601, Australia; website http://www.cambia.org/ or email [email protected]).
7. Other components of these vectors may be similarly protected by other parties. Obtaining licences for these components (if required) is the responsibility of the user.
8. The Recipient agrees to hold harmless the University, and any of its employees (past or present), agents and officers from any and all liabilities or claims resulting from transfer to, use, or storage of the Materials by the Recipient.
9. This Agreement cannot be assigned. 10. This Agreement is subject to Australian Law and exclusive interpretation by the Australian Courts.
I, (name)________________________________, an authorised officer of (institute) ______________________
___________________________________________________________________________________________,
have read and agree to be bound by the above terms on behalf of and for (institute) ________________________
___________________________________________________________________________________________.
Signed: ……………………………………………. Name: ………………………………….……………………
(Please print clearly) Position:………………………………………………………………………………. Date: ……………………
Xylanase Assay Kit
XYLANASE ASSAY PLASMID VECTOR KIT Please find enclosed the following plasmids as requested:
pUbiSXR
pActXYN
pUbiGUSPlus
Plasmids are spotted onto paper in a solution of 50% sucrose in TE buffer with loading dye. Re-suspend the blue spots in 5 –10 l of water or TE by pipetting back and forth several times. Transfer to a tube and dilute one in ten before transforming into E. coli cells. Full plasmid sequences with annotations can be found in GenBank under the following accession numbers:
AY452753 (pUbiSXR)
AY452735 (pActXYN)
AY452736 (pUbiGUSPlus)
Best wishes and good luck with your research!
ARC Centre of Excellence for Integrative Legume Research
John Hines Building (62) The University of Queensland St Lucia Brisbane, AUSTRALIA 4072 Ph: +61 7 3365 3550 Fax: +61 7 3365 3556 Email: [email protected]