ASU TVDC Progress Report
11/25/08
Kathryn F. Sykes and Stephen A. Johnston
Completed Milestones: 25, 26 and 32, 33, 34
Active Milestones: 28 and 35
Currently Inactive Milestones: 30, 36-38
Slide 1
MILESTONE 28
Build SCHU S4
proteome
Gray: (sub)milestone title
Red: inactive
Green: in progress
Build ORF expression
library corresponding
to proteome
Generate complete
protein-fragment library
Array protein-fragments
for T cell stimulation
assays
Complete
In process
Designing
Slide 2
Update on ORF-Library production
1.
2.
3.
4.
All ORFs that were not amplified previously due to
degradation of stored FTU primers have been
successfully amplified using the new primers.
There are total of 2,229 high quality linear expression
element constructs. of FTU ORFs. These are ready to
be used as template constructs for (IVT) polypeptide
library production.
The concentrations of these templates were
determined and amounts needed for IVT reactions
were calculated.
Appropriate volumes have been transferred to IVT
plates and dried to completion.
Slide 3
Linear expression templates of the
remaining ORFs
• Using the new
primers, 128 of
ORFs were
amplified from
genomic DNA
• The linear
expression
templates of these
ORFs were
successfully
assembled
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT DNA gels\Long ORF 5 Plate 2 and short 2 with new primers 11-7-08_
Slide 4
Initial large-scale batch of protein
production
• We used templates from the Long ORF2 plate
for the first large batch of IVT polypeptide
production.
• A total of 336 (4 of 96-well plates) polypeptides
were translated without 35Smet radiolabel.
• For the QC plate, we in vitro translated 12 ORF
constructs from each of these 4 x 96-well plates
(total of 48 ORFs) and 1 GFP template with
35Smet radiolabel for visualization and analysis.
Slide 5
First QC test
1. The GFP control was visualized; no
radioactive FTU proteins were seen.
This corresponds to the only template
not frozen and dried before performing
the IVT reactions.
2. We supposed that the templates might
have degraded during freeze-thaw cycle
or speed-vac drying.
Slide 6
QC test run #1
GFP
GFP template was not dried prior to IVT reaction
FTU samples were dried in a common speed-vac
(shared with chemistry group)
Slide 7
Troubleshooting templates
bp
2500
2000
1500
1000
500
1 2 3 4 5 6 7 8
• Samples in lanes 1-8 are
representative of 12
templates used in the IVT
reactions for QC plate
• The image shows that the
templates are not
degraded
• They should be sufficient
for IVT reactions
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT DNA gels\
Testing Lee Storage 11-12-08 Long ORF2 PCR plate 2 Row B1-B8 2
Slide 8
Testing LEE templates for IVT reaction
•
•
•
•
•
We chose 4 ORF templates that
were used for IVT reactions in
previous QC plate
The same templates were used
again in fresh IVT reactions with
radiolabel and the fractionated on
gel
Lanes 1-4: FTU templates w/o
drying
Lanes 5 and 6: GFP templates
dried in 2 different speed vacs
Conclusions: lyophilization is not
necessary, speed-vac can work
also-assuming no equipment
issues
1 2 3 4 5 6
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT 35S gels\F tularensis proteomic library\Long ORF 2\
Long ORF 2 Plate 2 Row B1-B4 and GFP 11-17-08 crop
Slide 9
Testing protein productions in library plates
KDa
125
100
75
50
37
25
20
15
10
• Silver stain gel of 4 bead
samples picked randomly
from the library plates
showed that proteins
were successfully
produced and efficiently
bound on beads
• The templates for these
IVT library plates were
transferred and dried in
different speed-vac than
used for QC plate #1.
• Synthesis was successful
for the library plates.
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT silver stain\LOng ORF2 B1 to B4 11-12-08 crop
Slide 10
Second large batch of IVT productions
100
150
50
75
50
37
25
20
15
10
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT 35S gels\F tularensis proteomic library\Long ORF 5\
Long ORF 5QC 11-21-08 2
• Autoradiograph of QC plate of 24 templates from Long
ORF 5 plate (lane 25: GFP as positive control)
• There are 144 ORFs in Long ORF 5 plates
• The average amount of proteins bound on beads is 10.2
ug per reaction
Slide 11
Conclusion
1. We have completed production of 480
FTU polypeptides
2. Another 236 FTU samples are in
process.
3. We plan to have the library completed by
the end of this month
4. We will start re-array the polypeptides
into pools next month
Slide 12
MILESTONE 35
Array hybridations with mouse RNAs
from virulent Schu 4 infection
& RT PCR confirmation of candidates
Gray: (sub )milestone title
Red: completed
Green: in progress
Virulent Schu 4 Samples
RT-PCR Confirmations
Initial samples
Dose-Response of Infection
Ongoing
Slide 13
Previous Status
• LAPT analyses have been performed on…
Two Dose Response Challenge Samples
103, 104, 105, 106, 107
101, 102, 103, 104, 105, 106, 107
1,3,5,7, and 24 hours post challenge
Samples from Ftc-64,5 received
• Intermittent LAPT failures over the past 6 weeks
• We have isolated the problem to the template switch primer
• New primes have been ordered and received.
• qPCR established using 6 genes and reconstitution
samples
• Questions have been raised about relative quantification
gene for test samples from dose-response experiments
Slide 14
Mean Cycle Threshold (Ct) Values for
qPCR Analysis of Reconstitution cDNA Samples
Input RNA
ng/ml
FTT0548 FTT0901 FTT0721
FTT0058
FTT1712c
1000
19.64
18.59
17.86
15.98
11.37
100
24.12
19.96
19.55
19.46
13.09
10
26.34
24.3
23.48
22.97
16.9
1
30.61
28.29
26.7
26.05
20
0.1
ND
30.07
30.01
29.76
23.58
0.01
ND
34.93
32.66
31.68
26.89
0.001
ND
ND
ND
ND
30.09
0
ND
ND
ND
ND
ND
Slide 15
Relative Expression of iglC to MutS
Sample
Ct SYBR MutS
Exp. 1 105
Ct SYBR
1712c
28.24
34.02
Expr. Level
SYBR
0.201
Exp. 1 106
25.23
32.27
0.484
Exp. 1 107
20.80
28.89
No Data
Used as
calibrator
Sample
Ct SYBR MutS
Exp. 2 104
Ct SYBR
1712c
31.39
34.65
Expr. Level
SYBR
0.102
Exp. 2 105
28.84
33.08
0.201
Exp. 2 106
25.70
31.28
0.510
Exp. 2 107
21.71
28.26
No Data
Used as
calibrator
Slide 16
Bacterial Load in First Time Course Experiment
Harvest Time Bacterial Load
1h
144
3 h
256
5 h
330
7 h
608
24 h
83,000
Slide 17
Upcoming Transcriptome Goals
• LAPT
• Evaluate presence of inhibitor in current
sample set
• Re-establish amplifications with reconstitution
samples
• Q-PCR validation of the hits
• New samples?
Slide 18
Action Items
• Terry wants beads in RPMI with antibiotics and will
convey this to Alex.
• Terry will send depositions from dose/response curves to
Mitch.
• Mitch will perform qPCR with the 24hrs sample to
resolve sensitivity question.
• Mitch will communicate with UNM regarding the LAPT
working again, with reconstitution samples for testing at
0.1 and 0.01.
Slide 19