UNM TVDC: ASU - UNM Tech Call Minutes 02/27/07
Prepared 2/27/07: Mindy Tyson/Barbara Griffith
Sent to ASU: 2/27/07
Edited by: Kathy, Mitch, Stephen (add dates)
Sent to NIAID: Barbara will add date
Present: Kathy Sykes, Mitch Magee, Barbara Griffith, Rick Lyons, Joe Breen, Stephen Johnston,
Marlene Hammer,
Absent: Kristin DeBord, Freyja Lynn, Vicki Pierson, Alex Borovkov
Action Item from previous meetings:
1. Mitch will send Barbara an example of probe QC verifications for the thermal cyclers- trying to
get from installer; 12/20- still trying to get documentation from the installer (NOTE: Barbara and
Mitch discussed alternate QC verification methods on 2/27/07)
Action Items from 2/27/07 meeting:
1. Stephen: will show Kathy how to quantitate the S35 protein to determine the protein mass
yield.
2. Kathy will estimate the additional cost if a feeding system is used to produce the proteins
3. Mitch will call Barbara on 2/27/07 regarding milestone timeline updates and 1/30/07 meeting
minutes (completed on 2/27/07)
4. Rick will send pdf of Ann DeGroot paper to ASU, Marlene, Vicki and Joe and Barbara will add
this topic to the agenda for the 2/28 Prime Tech call.
5. Note added after call: NIH forwarded ASU request to TIGR and ASU should hear from TIGR
within a few weeks of 2/27/07
Discussion of the Progress of Milestones
A. Active/Inactive Milestones:
a. Milestones – Sykes
i. Completed 25
ii. Active – 26, 28
iii. Inactive – 30
b. Milestones – Johnston
i. Completed 32
ii. Active – 33, 34
iii. Inactive – 35-38
B. Milestone 26:
a. Flow Chart
i. Prepare a high-throughput protein production system
1. Test ORF synthesis and select expression constructs
a. Modified ORF expression templates are designed as
needed for IVT and purification step optimizations.
2. Select and test IVT Protocols
a. Alternative in vitro protein production systems are being
evaluated.
3. Select and test protein purification protocols
a. StrepAvidin and Nickel based purification methods are
being prepared.
b. Current Data
i. Question:
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1. Can our readout of in vitro protein production be improved by
switching away from tRNA-fluorescent detection to amino acidradiolabeling?
a. Expression Template (see image, slide 5)
b. For comparison: Fluorescent tRNA-labeling (see imageslide 6)
i. Lysate alone shows innate fluorescent protein
only
c. 35S-radiolabeled IVT reaction (see image, slide 7)
i. Lane 2 is lysates w/o template and shows no
radioactive signal indicating no nascent protein
ii. Lane 1 is lysate w GFP template and shows
dominant GFP protein produced
ii. Conclusion:
1. 35S-methione labeling of in vitro translated products provides
greater specificity of detection.
iii. Problem/troubleshooting
1. Yields obtained from test translation reactions appear to be
generally lower than desired and modestly lower than that of
circular control templates. (Using labeling as a relative measure,
we roughly estimated our yields to be ~10-50% of controls.
Controls are advertised as ~1-5 ug.)
2. Rather than attempt protein purification from low yield reactions,
yield improvement was pursued next
iv. Question:
1. Can we improve product yields with:
a. Two different feeding systems- wheat germ or a mix and
match system with Invitrogen and Roche reagents
b. Concerned that unfed, single ivt reactions give rise to too
little product
c. Sales representatives predict 1-5 ug/rxn though some
literature citations indicate up to 10-15 ug are possible
d. ASU is currently quantitating by comparison to control
template protein production level.
e. ASU prefers not to run multiple reactions and is trying to
produce the most protein from a one reaction.
f. Lysate “feeding” system option:
i. Standard IVTs do not yield sufficient quantities
of protein to generate target amount of 10 to 15
ug final purified material for T cell assays
g. Wheat germ translation lysate option:
i. This is a feeding system designed especially for
the plant system
2. RTS continuous-exchange cell-free technology (CECF)
increases yields (see image , slide 11)
a. Diagram of the feeding system
b. Don’t need to add volume to increase reagents, as it is a
dialysis system
3. Time course of protein production (for Supercoiled plasmid) (see
image, slide 12)
a. Compares traditional coupled ivt to RTS using the
CECF lysate feeding system
b. Traditional plateaus at 4 hrs and CECF plateaus at 6-8
hrs
c. CECF shows much higher protein yield
4. LEE template in In vitro “feeding” reactions (see image, slide 13)
2
a. Lanes 2 and 8: compare supercoiled plasmid in lane 2
and linear in lane 8
b. See plateau after 2 additions of the template
c. Feeding in: more template
d. Can make circular and linear template perform
identically, if give system more linear template. Perhaps
the linear template gets degraded over several hours
e. 1 ug of template added multiple times is easy and
feasible for the linear DNA.
f. Slide 13: only adding more DNA template
g. ASU didn’t show the data but stated that adding more
enzyme produces even more product
h. Won’t use Wheat germ system, because it produced
much less product than Invitrogen or Roche E.coli
systems
i. Slide 15: lane 13… there was no increase in template in
any of these lanes. Feeding means in feeding system
but no increase in template. Just lane 16 is adding
enzyme. Lane 13 linear FTU has no product while lane
12 shows more product. 3 hr reactions due to plateau of
reagent.
j. Joe: how much product is in lane 12 on slide 15?
k. Kathy: reps say 1-5 ug. We are moving up but don’t
know level reached. S35 will allow us to follow the
reactions and purified product will allow us to quantitate
using the luminex and antibody
l. Joe: can you get to 10 ug then?
m. Kathy: based on TCA counts- are 2-4 fold increase (2
fold with more template and 2 fold more with more
enzyme)
n. Stephen: will show Kathy how to quantitate the S35 to
protein mass.
o. Roche ; Lane 15 and 16: 16 has more enzyme and TCA
counts show double the radioactivity, though eye can’t
tell
p. Comfortable with linear system
q. Joe: temperature performed?
r. Kathy: 30 C
s. Stephen: how much cost will this add?
t. Kathy: don’t know yet
u. Didn’t do purification and will quantitate after purification
v.
v. Conclusions:
1. Use of linear templates in extended IVT reactions requires
addition of fresh template.
2. Yield of the IVT reaction with four sequential additions of linear
template is similar to that from a circular template.
3. Supplemented IVT reactions may be limited by RNA polymerase.
vi. Determining optimal IVT format
1. Comparison of Wheat germ, E. coli (Invitrogen) and E.coli
(Roche)
a. Slide 15: lane 13… there was no increase in template in
any of these lanes. Feeding means using the feeding
system but no increase in template was fed into the
system. Just lane 16 is adding enzyme into the feeding
system. Lane 13 linear FTU has no product while lane
3
12 circular FTU shows more product. These are 3 hr
reactions due to plateau of reagent.
b. Joe: how much product is in lane 12 on slide 15?
c. Kathy: Sales representatives say 1-5 ug. ASU is
increasing the yield but don’t know yield reached. S35
will allow us to follow the reactions and purified product
will allow us to quantitate using the luminex and antibody
d. Joe: can you produce 10 ug with feeds of template and
enzyme?
e. Kathy: Based on TCA counts, the yields are 2-4 fold
increased (2 fold with more template and 2 fold more
with more enzyme). Lane 15 and 16: 16 has more
enzyme and TCA counts show double the radioactivity of
lane 15, though eye can’t detect the 2 fold increase.
Didn’t do purification and will quantitate after purification
f. Action: Stephen: will show Kathy how to quantitate the
S35 protein to determine the protein mass yield.
g. Comfortable with linear system
h. Joe: What temperature performed is used?
i. Kathy: 30 C
j. Stephen: how much cost will this feeding add to the
protein production?
k. Kathy: don’t know yet
l. Action: Kathy will estimate the additional cost if a feeding
system is used to produce the proteins
2. Top IVT Format to date
a. E. coli lysate and buffer from Invitrogen.
b. Dialysis feeding system from Roche.
c. Linear expression template. With feeding, yields are
comparable to plasmid-based reactions.
d. Feeding for 12 hrs with supplements of the DNA
template and T7 RNA polymerase
vii. New BirA recognition sequence
1. In E. coli, the biotin protein ligase (BirA) transfers activated biotin
to the ε-amino group of a specific lysine residue of the biotin
carboxyl carrier protein (BCCP) subunit of acetyl-CoA
carboxylase. Sequences of naturally occurring biotinylated
substrates are highly conserved across evolutionary boundaries.
Cross-species biotinylation has been demonstrated in several
systems.
2. However the new BirA site does not resemble the consensus
sequence of any naturally occurring biotinylation sites.
3. High efficiency BirA site (see image, slide 18)
4. Expression Template (see image, slide 19) – can add ubiquitin
C. Milestone 28:
a. Flow Chart:
i. Build SCHU S4 proteome
1. Build ORF expression library corresponding to proteome
a. Just building the ORFs which will be put into the ivt
template thereafter
b. Gene specific oligos have been master-arrayed
2. Generate complete protein-fragment library - Inactive
3. Array protein-fragments into measurable pools for T cell
stimulation – Inactive
b. Master plate Arraying (slide 21)
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i. Our electronic management system, GEMS, has designed and then
instructed our robot to normalize concentration of all oligos, pool forward
and reverse gene specific primers for each ORF, then identify pairs
designated for PCR-amplifying natural sequence ORFs from genomic
DNA. The remaining primers will be used as part of the gene building
protocol to improve the ORF.
ii. Some ORFs will be direct from the genome and others will be
synthetically built to improved them.
iii. Green colored wells represent the forward and reverse primer pairs
being cherry-picked into a master plate 384-well plate for wildtype/natural
sequence production of an ORF and put it into 384 well master plate for
high throughtput PCR production. Should have no trouble making this
set of proteins.
iv. The ORFs from the red colored wells will be synthetically built. This
master plate arraying is designed, programmed into the robot, and then
the robotic performance is entered into the electronic management.
c. Work plan for upcoming month:
i. Production of wild type ORFs will begin.
ii. High efficiency biotin attachment peptide will be tested within template in
co-translation/biotinylation reactions.
iii. Strepavidin purifications of protein-fragments will be tested. This option
has the advantage of super high affinity.
iv. Nickel purification will be conducted in parallel to allow comparison of
scheme efficiencies and purities. The Nickel –His has lower affinity but it
is incorporated into the protein. It can be hidden in protein folds, which is
a disadvantage.
v. Yield and solubility of pilot set of the SCHU S4 protein-fragments will be
assessed by measuring purified, antibody-bound protein on Luminex.
B. Milestone 32:
a. Flow Chart
i. Array Oligo List refined, 70mers oligos procured, GDP-Oligos designed
and procured
1. 70 mer Oligos designed for array
a. 1804 probes designed - completed
b. Synthesis complete - completed
c. Oligos received - completed
i. Design and production of LVS-specific probes –
in progress; will be ordered this week and can
be added to the microarray later.
2. GDP primers
a. 183, 7 mer primers designed - completed
b. Synthesis complete - completed
c. Primers received – completed
b. Microarray Printing Action Plan Update
i. PE Spotarray
1. Mechanical control in Z-axis was problematic
a. Machine sent to factory and returned to ASU on 2/23/07
b. Initial verification of motion will be completed this weekwatched for 3 days continuously to assure no arm
failure. Will be testing more with real printing material.
ii. Nanoprint problems
1. Sonicator system
a. Redesigned and refabricated
b. Installation on 3/2/2007
iii. Full deck run completed: 57 slides per full deck print
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1. Full set of 1804 probes representing the whole genome and
ribosomal controls
2. 15 Corning Ultragaps/32 Poly-L-Lysine printed
3. Waiting on TIGR review of application for PFGRC slide
availability – sent reminder to Kimberly Stempke at NIH
4. Action:Joe: will check on the TIGR status for ASU
5. Note added after call: NIH forwarded ASU request to TIGR and
ASU should hear from TIGR within a few weeks. Joe may not
need to follow up afterall.
C. Milestone 33:
a. Flow Chart:
i. Printing and testing GDP Confirmed
1. Printing arrays
a. Comparions of substrate
b. Poly-L Lysine vs Corning Ultragaps
c. Compare TIGR PFGR Arrays to in house arrays
2. GDP Confirmation
a. RNA shipped 1/29/2007, analyzed, and initial
hybridizations done
b. Testing of linear amplification of procaryotic transcripts
(LAPT)
b. Initial Hybridization Screens
i. 10 ug RNA converted to cDNA using random primers and labeled with
Alexa555, hybridization on slides for 16-18 hours, washed, and scanned
ii. Doing single color arrays now and will use genomic DNA later as the
normalizing target.
iii. Two separate preparations of RNA from UNM for both SCHUS4 and LVS
and two independent full-gene print runs. These are hybridized to
separate slides since currently using a single color system.
iv. Images acquired and spotfinding performed with GenePix Pro
v. Data analysis using GeneSpring GX
vi. Combined data (all substrates) looking for genes up or down regulated
between LVS and SCHU S4 > 3 fold and significant between reps.
vii. 61/1804 genes are down-regulated comparing SCHU S4 to LVS
viii. 26/1804 genes are up-regulated comparing SCHU S4 to LVS
ix. Afterwards: ASU pooled all LVS RNA and pooled all SCHU S4 RNA to
have common pool of approximately 160 ug each of LVS RNA and
SCHU S4 RNA.
c. Differentially Expressed Genes SCHU S4 to LVS (3 Fold up and down and
significant)
i. Many are hypothetical genes (slide 27)
ii. Blue colors are downregulated and red colors are upregulated
iii. Top panel shows all 81 genes
iv. Hard to put into KEGs or othologs.
d. Should have infected organ RNAs from UNM in another 2-3 weeks
i. ASU will be soon be ready for the RNA from UNM soon
D. Milestone 34:
a. Flow Chart
i. Pilot studies of optimization of RNA isolation and hybridization conditions
1. RNA Isolation (UNM)- will send within 2-3 weeks
a. Initial testing of heavily infected lungs
b. Perform CFU analyses and compare with purified RNA
2. Hybridization Conditions
a. Testing Maui Hybridization chamber
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b. Amplification testing of Schu S4 RNA with and without
mouse lung RNA
b. Work plan for upcoming month: Transcriptomic approach
i. Order LVS unique probes and GDP primers
ii. Compare hybridization of ASU array to TIGR microarrays.
iii. Perform additional testing of poly-l-lysine and Corning ultragap slides
from SCHU S4 RNA labeled in the presence of mouse lung RNA
iv. Perform GDP amplification of purified SCHU S4 RNA in dilution series
alone and in the presence of normal mouse lung RNA
Next ASU Tech Call:
ASU Tech call: March 27, 2007 Tuesday noon-1pm MT (2-3pm ET)
Administrative:
Action: Mitch will call Barbara on 2/27/07 regarding timeline updates and 1/30/07 meeting
minutes (completed on 2/27/07)
Publication- Rick described paper in Vaccine from Anny De Groot
 Anny De Groot (at Brown and Epivax): has funding to look at antigens from Martha’
Vineyard patients and has developed list of candidate proteins. Could UNM test some of
the proteins as outside candidates identified by other means, within the scope of
contract? Rick recalls writing this option into the contract.

Action: Rick will send pdf of Ann DeGroot paper to ASU, Marlene, Vicki and Joe and will
add this topic to the agenda for the 2/28 Prime Tech call.

Marlene is concerned about intellectual property

Stephen: would we test the epitopes themselves or the serum?

Rick: Ann was not clear on this topic. She used bioinformatic approach to identify
potential peptides and looked at secreted and for the most common HLA antigens in
Caucasian populations; She looked a promiscuous peptides that would target the B54,
A27, B7. She found 27 stimulated Interferon gamma production in >95% of the exposed
population by performing ELIspots on peripheral blood cells. Rick doesn’t know if she is
examining a pool of these peptides or the genes. She is trying to use some of these as
vaccines in the model, assuming the LVS model, but it is unclear in the paper. Ann
communicated to Rick that they were able to demonstrate protection with some of the
peptide candidates, though this is not stated in the paper.

Stephen: Could immunosignature the sera if Anny wants?

Rick: Thinks sera may be hard to obtain from Anny.
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