ASU TVDC Progress Report
8/26/08
Kathryn F. Sykes and Stephen A. Johnston
Completed Milestones: 25 and 32, 33, 34
Active Milestones: 26, 28, 35
Currently Inactive Milestones: 30, 36-38
Slide 1
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
To Be Determined
Slide 2
Previous Status
• Initial qPCR validation studies revealed good results for
FTT901
• LAPT analyses have been performed on the original time
course samples (1,3,5,7, and 24 hours post challenge)
Slide 3
Targeted genes for qPCR
FTT Designation
Gene symbol
Gene Name
FTT0901
lpnA (Tul4)
conserved hypothetical lipoprotein
FTT0721c
katG
Peroxidase/catalase
FTT1712c
iglC2
intracellular growth locus, subunit C
FTT0548
dnaQ
DNA polymerase III, epsilon subunit
FTT0058
atpB
ATP synthase subunit
FTT0256c
Lipopolysaccharide protein
Slide 4
Validation of qPCR Primers
(Representative graphs)
FTT0721
FTT0058
Melting Curve
Quantitation
0.001
0.01
0.1
0.001
0.01
0.1
Slide 5
Reconstitution Samples Assessed by qPCR for FTT0721
Melting Curve
Amplification Plot
Genomic DNA Dark Blue
cDNA Light Blue
Negative Pull Down Peaks
Standard Curve
0.01
0.1
Slide 6
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
ND = not detected
Slide 7
Effect of Plate Style on Melting Temperature Profile
White Wall Plate
FTT0058
FTT0901
Regular Plate
Slide 8
Melting Temperature Shift in Reconstitution Samples
is not the Result of Secondary PCR Products
Input RNA in Nanograms
NML
100
10
1
0.1
0.01
0.001
Slide 9
Conclusions
• We have validated 6 qPCR primer sets with
genomic DNA
• Reconstitution samples (SCHU S4 RNA
diluted into normal mouse lung RNA) have
been assayed by the qPCR system
• With 4 genes we could determine Ct values
down to 0.01 ng/ml SCHU S4.
Slide 10
Upcoming Transcriptome Goals
• Q-PCR validation of the hits
•Design and validate genes for normalization
of qPCR results
•Run qPCR on the original time course
samples
•Time Course Experiment Repeat
•Repeat LAPT of first experiment
•Rat and Mice
•Challenge with 104 SCHU S4 organisms
•Harvest 1,3,5,7 and 24 hours
•Parallel cultures in Chamberlain’s medium
Slide 11
MILESTONE 26
Prepare a highthroughput protein
production system
Gray: (sub )milestone title
Red: completed or inactive
Green: in progress
Test ORF synthesis
and select expression
constructs
Select and test
IVT Protocols
Select and test protein
purification protocols
Expression templates
for prokaryotic expression
further optimized
High yield IVT protocols
further optimized
Purification strategies have
been identified
Slide 12
26.1 Last report
New designs enable size of LEEs to
reach to 1500bp
Slide 13
Long LEE assemblies in HTP format
MS26.2 Last report
• Large LEE cassettes can be
successfully used in IVT reactions
• Proteins with MW up to 50Kd (~ 500
amino acids in lengths) can be
produced in vitro
Slide 15
26.2 Update
Slide 16
IVT Product Analysis
Invitrogen
Silver stain
New England Lab
autorad
Invitrogen
New England Lab
250
150
100
75
50
37
25
20
15
10
1,2,3:No template, ova, and FTU901 Invitrogen bead supernatant (unbound products).
4,5,6: No template, ova, and FTU 901 NEB w/o bead (whole reaction).
7,8,9: No template, ova, and FTU 901 NEB bead supernatant (unbound products).
10,11,12: No template, ova, and FTU 901, NEB bead eluant (bead bound and eluted products)
MS26-3. Last report
Slide 18
Pilot Testing of Trx-IVT
samples on NHP IFN-gamma
response assay at UNM
Slide 19
Status
• Antigen stimulation using polypeptides
attached to anti-Trx beads significantly
decreases amount of cross-reactivity in the
NHP immune assay.
• The NEB IVT mix displays significantly less
cross-stimulation of the NHP immune cells as
compared to the full E. coli lysates.
Slide 20
MS26-3. Update
Slide 21
Frozen NHP T-cell assays: Invitrogen vs. NEB
generated polypeptides
1:10
Neat
1:10
Neat
Ova
FTU901
No template
Invitrogen IVT
bead bound
samples
NEB IVT bead
bound
samples
Cost analysis of FTU proteome
Cost analysis of IVT reagents for FTU contract
1 Rxn
Long ORF (2500 rxn)
Short ORF (4500 rxn)
With 10% discount
NEB (25ul rxn)
$20.00
$50,000.00
$90,000.00
aTrx antibody 10ul/rxn beads
$1.97
$4,925.00
$8,865.00
aTrx antibody 50ul/rxn beads
$9.84
$24,600.00
$44,280.00
M280 Tosyl Beads (30mg/ml)
$1.76
$4,400.00
$7,920.00
MyOne Carboxy Beads (10mg/ml)
$0.88
$2,206.25
$3,971.25
$33.60
$79,000.00
$142,200.00
With Discount (30%)
Total cost NEB (25ul), 50ul beads, Tosyl
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
into measurable pools
For T cell stimulation
Inactive
Preparing plan
Inactive
Slide 24
Decisions Made 8/26/08
Since Barbara was absent on 8/26, official minutes were not taken. On 9/4/08, ASU provided the following bulleted
items summarizing decisions made in the last ASU TVDC call (8/26).
1. Antigen stimulation using polypeptides attached to anti-Trx beads significantly decreases amount of cross-reactivity
in the NHP immune assay.
2. The NEB IVT mix displays significantly less cross-stimulation of the NHP immune cells as compared to the full E. coli
lysates.
3. These 2 results hold even when using the frozen NHP lymph node cells.
4. By combining the use of the NEB pure mix with the Trx purification protocol, we are able to almost completely
eliminate all cross-reacting material from the T cell assay.
5. All participants- including Rick, Joe, and Marlene- agreed that the scheme for proteome production is fully developed
and full-scale library production should begin at ASU.
6. This concludes ML26.
Slide 25