Transcript ASU TVDC 12.17.08 minutes final

ASU TVDC Progress Report
12/17/08
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
Complete
In process
Slide 2
Autoradiograph of QC plates
LONG 1
LONG 3
LONG 4
QC plate description
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There are a total 8 QC plates
BAG (build a gene)
Short 1
Short 2
Long 1
Long 2* ( 1st QC plate failed but the library samples were successfully translated as
showed in last report)
Long 3
Long 4
Long 5
SDS gels and autoradiograph if these QC plates shown 96% of templates are successfully
translated.
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Autoradiograph images are stored at:
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT 35S gels\F tularensis
proteomic library
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SDS Coomassie stained gel images are stored at:
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT Coomassie gels\F
tularensis Library
Overview of milestone 28
• We successfully synthesized and purified
microgram quantities of 2, 229 polypeptides
corresponding to the F.tularensis SCHU4
proteome.
• These proteins will be pooled into groups of 7
polypeptides for immune assays.
• The arraying scheme will be completed by the
end of this week.
• These F.tularensis antigen pools will be sent to
UNM in January 2009.
Pooling strategy description
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Bead bound purified polypeptides are currently each
stored in 100 ul PBS.
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Polypeptides will be pooled by columns
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Each of these samples corresponds to one IVT reaction.
Each will be split :50 ul will be used for pooling, 50ul will be
stored for next experiment set.
there are 7 rows per a column.
each pool will contain 7 different polypeptides
Each 96-well plate will generate 12 pools (from 12 columns)
Total volume of each pool will be 350 ul
We will add 10 ul of PBS into each pool to make a total
pool volume of 360 ul for distribution.
Proposed diagram for pool
distribution
Starting volume:
360 ul per pool
180 ul for
Lymph cell assays
90 ul for
ELISpot assay
90 ul for
ELISpot assay
-duplicate
180 ul for
PBMC assays
90 ul for
ELISpot assay
90 ul for
ELISpot assay
-duplicate
Upcoming Proteome Goals
Deliver library SAFELY
Work with Terry on any issues during
assays
Prepare to produce proteins recombinantly
as needed for TVDC scientists
Slide 8
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 9
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 (Rat time course) received and purified
• Intermittent LAPT failures over the past 6 weeks
• New Primer worked in two consecutive experiments and the dose in
an expected dose response range.
• qPCR established using 6 genes and reconstitution
samples
Slide 10
qPCR Amplification Plot – Dose Response
Slide 11
qPCR Amplification Plot – 83,000 - Diluted
Slide 12
Bioanalyzer Results
Slide 13
Upcoming Transcriptome Goals
• LAPT
• Process Mouse and Rat time courses
• Q-PCR validation of the hits
• Repeat MutS and iglC relative quantifications
in samples with defined bacterial loads
• Preliminary results indicate that we will need
at least 2,000 bacteria per lung per time point
Slide 14
Action Items
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Terry-has Terry sent the correct plates to Alex and communicated on best
plates and media for the beads/polypeptides storage prior to shipment to
UNM? ASU needs the correct plates and will have the beads/polypeptides
in the correct media for Terry.
Terry: please discuss freezing the bead slurry with Alex at ASU. Is ASU’s
freezing plan okay with UNM?
Rick/Terry- after ASU generates the LAPT results, UNM can send another
batch of mouse RNAs as needed by ASU, for repeating the time course
experiment. Rats are infected with higher doses already so won’t need to
repeat the rat experiments, but mice were infected with lower doses, which
may be below the qPCR lower limits of detection.
Slide 15