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 characterised
Excellent assay systems
available
Qualitative, quantitative
Many reports of endogenous
GUS-like activity
Problems with cell autonomy
Quenching
LUC
(luc)
Luciferase
(firefly/
bacterial)
Luminescence
Extremely sensitive
Qualitative, quantitative
Very expensive
Very labile
Cannot increase sensitivity
antRC
Regulatory
proteins
Anthocyanin
pigment
Cell-autonomous
No substrate required
Non-destructive
Not for quantification
Toxicity problems
GFP
(gfp)
Green
fluorescent
protein
Fluorescence
Cell-autonomous
Non-destructive
No substrate required
Pictures look great!
Problems with quantification
Chlorophyll autofluorescence
(light
emission)
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
1m gold
particles
Plasmids containing
construct and
reporter
Quick, cheap, easy
Focus Counting: Threshold Effect
Actin
E
X
S M
D1
D3
B
Barley
Bar l ey
GFP
Wheat
Wheat
OG
UTR
TSS
OG X
OG S
OG M
OG D1
OG D3
1200 bp
900
600
300
0 bp
0
0.5
GFP:GUS
1
1.5
Focus Counting: Threshold Effect
80
Focus Intensity (RLU)
70
60
50
40
Saturation
threshold
30
Detection
threshold
20
10
0
P1
P2
P3
P4
P5
P6
P7
Focus Intensit
Focus Counting – Threshold Effect
1
2
4
3
5
6
Promoter
B
80
Average Focus Insensity
Number of Foci
40
20
0
1
2
4
3
Promoter
5
6
Number of Foci
Focus Intensity
60
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:
Xylanase
•
•
•
•
•
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
Physical Optima
Temperature Response @ pH = 4.5
• pH = 4.5
4
4
Absorbance @ 590 nm
Absorbance (590 nm)
pH Response @ 40 C
3
2
1
0
3
• Temp. = 40C
2
1
0
3.0 3.5 4.0
4.5 5.0 5.5 6.0 6.5 7.0
7.5 8.0 8.5
0
20
pH
80
4
3
2
2
R = 0.9924
1
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1
Substrate Concentration
Absorbance @ 590 nm
Absorbance @ 590 nm
60
Temperature (Degrees C)
Enzyme Concentration
Extract Dilution
40
• Linear response
to enzyme
concentration
• Substrate
saturates at 0.5%
2.5
2.0
1.5
1.0
0.5
0.0
0
0.5
1
1.5
AZCL-Xylan (%)
2
2.5
Time Response
• Linear over
time
Absorbance (A570)
Time Response - P/100
0.80
• Up to 48 hr
0.60
0.40
y = 0.0004x + 0.0377
R2 = 0.9909
0.20
0.00
0
500
1000
Time (min)
1500
2000
• 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
E
X
S M
D1
D3
B
GFP
UTR
Actin
Actin
Barley
Bar l ey
Wheat
Wheat
OG
OG
TSS
OG X
OG X
OG S S
OG
OG M M
OG
OG D1
OG
D1
OG D3
OG
D3
1200 bp
900
600
300
0 bp
0
0.5
GFP:GUS
1
1.5
Deletion Analysis – GUS:XYN
GUSPlus :Xylanase
100
Hordein
Globulin
Glo X
Glo S
Glo M
Glo D1
Glo D2
Glo D3
Negative
1200 bp
900
600
300
0 bp
200
300
400
500
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 Assays
in Plant Tissues
Claudia Vickers [claudia.vickers@csiro.au]
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.
Introduction
Reporter Genes Used
Detailed Method
Solutions
References
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Introduction
Quantification 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
MATERIAL TRANSFER AGREEMENT (MTA)
ARC Centre of Excellence for
Integrative Legume Research
John Hines Building (62)
The University of Queensland
St Lucia
Brisbane, AUSTRALIA 4072
Ph:
Fax:
Email:
+61 7 3365 3550
+61 7 3365 3556
director.cilr@uq.edu.au
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: ______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
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 rights. 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 vectors@cambia.org).
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.
Phone:
__________________________________
Fax:
__________________________________
___________________________________________________________________________________________,
Email:
__________________________________
have read and agree to be bound by the above terms on behalf of and for (institute) ________________________
I, (name)________________________________, an authorised officer of (institute) ______________________
___________________________________________________________________________________________.
Signed: ……………………………………………. Name: ………………………………….……………………
(Please print clearly)
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.
Position:……………………………………………………………………………….
Date: ……………………
Xylanase Assay Kit
ARC Centre of Excellence for
Integrative Legume Research
John Hines Building (62)
The University of Queensland
St Lucia
Brisbane, AUSTRALIA 4072
Ph:
Fax:
Email:
+61 7 3365 3550
+61 7 3365 3556
director.cilr@uq.edu.au
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!
Thanks
• Supervisors
– Gangping Xue (CSIRO)
– Peter Gresshoff (UQ)
• Scholarship
– Grains Research and Development Corporation