UNM TVDC: ASU - UNM Tech Call Minutes 10/24/06
Prepared 10/25/06: Barbara Griffith
Revised 10/27/06:: Kathy Sykes and Mitch Magee
Sent to NIAID : 10/27/2006
Present: Kathy Sykes, Mitch Magee, Marlene Hammer, Mindy Tyson, Barbara Griffith, Rick
Lyons, Joe Breen
Absent: Kristin DeBord, Vicki Pierson, Freyja Lynn, Stephen Johnston
Action Items:
Mitch will send Barbara an example of probe QC verifications for the thermal cyclers
Rick, Terry, Kathy, Barbara, Cheryl - will discuss the peptide arraying at Woods Hole meeting.
Mitch- tell UNM what RNA samples are needed from test tissues,
Mitch- have list of expensive reagents and vendors for Barbara on 10/26.
Barbara will call Mitch at 9 am ASU time and 10 am MT on Thursday 10/26.
Kathy prepares the proteomics reagents presentation for Woods Hole.
Barbara- will ask Kathy to show their results and QC on the uncoupled transcription/translation
reactions at the 11/21/06 ASU tech call (Note- request added after the 10/25 Prime Tech Call)
Barbara- will ask Mitch to show their hybridization results at the 11/21/06 ASU Tech call (Noterequest added after the 10/25 prime tech call)
Semi Annual Quality Assurance Report
1. ASU- provided QA schedule for microarray spotters, microarray reader, pipettemen, freezers,
refrigerators, and safety/eye washes
2. The new Hygro culturing system is under warranty for the next year.
3. The thermal cyclers are new and also under warranty. There is a printout of each temperature
4. Action: Mitch will send Barbara an example of probe QC verifications.
5. Microarray spotters and printers are under maintenance agreements
6. ASU has a web based Rhees monitoring system for freezers and refrigerators which
autocommunicates whenever a temperature is out of range. It has a system of call downs.
Discussion of the Progress of Milestones
A. Active/Inactive Milestones:
1. Sykes – 25 completed; 26 active; 28-30 inactive
2. Johnston – 32 completed; 33-34 active; 35-38 inactive
B.
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Milestone 25: Design protein-fragment library based on SCHU S4 sequence
Computer program for oligo prediction is complete
500 peptides to be arrayed and used in Tcell assay workup have been received.:
Question: Rick and Kathy need to discuss the format for arraying the peptides, including
peptide concentrations. What is the limitation of the peptide concentration and cellular
response? This pilot set will help us. UNM is vaccinating primates and mice to get some
good substrates.
Question: Rick- arraying into what volume and in pools of some complexity up front?
K: arraying= how they will organize on a microtiter plate.
Kathy plans to setup a range of complexities for the T cell assay and would set up from
zero complexity to much more complexity. What happens with 10 peptides at various
concentrations or up to 50 at various concentrations? Kathy thinks many options will be
tested .
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Rick: will we array in a microtiter plates or going smaller if we can, perhaps on slides?
Titration of the peptides in the T cell assay itself. How few cells and how little peptides
are needed per assay?
Rick: can the peptides be sorted and be sterile? Alternatively , ASU could send UNM the
peptides, though ASU has more robotic experience. Cheryl: in context of ELI spotsreconstitute in DMSO in the hood and nothing will grow in DMSO Rick: really needs the
peptides clean – rather than truly sterile.
Action: Rick, Terry, Kathy, Barbara, Cheryl - will discuss the peptide arraying at Woods
Hole meeting. Team needs to discuss complexity and titration issues. Call Cheryl in on
a cell phone too as needed.
C. Milestone 26: Design protein-fragment library based on SCHU S4 sequence;
Confirmation of gene expression (Design HTP SOPs, Test HTP SOPs, ORF library
production, confirm gene expression).
1. Transcription/Translation Components for Linear Expression Elements (LEEs)
i. Promoter Elements
1. T7 promoter-6x His-Biotin acceptor site-Ubiquitin (386 bp)
2. T7 promoter-6x His-Biotin acceptor site (161 bp)
ii. Termination Element
1. HA tag-T7 terminator (158 bp)
2. Assembling of promoter, ORFs , and term has been optimized.
3. Templates have been tested in in vitro transcription/translation versus in vitro
transcription->translation systems.
4. Products are detected and systems compared.
Method for Expression Construct Assembly
• Synthetically build promoter and terminator elements, optimizing in terms of cost and
time. Also optimizing the whole expression construct for expression, translation and
purification.
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Synthetically build or amplify Schu4 open reading frames (ORFs) as per SynBuild,
which is a new software program.
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Construct LEEs using optimized promoter, ORF, and terminator elements. ASU has
made the optimal promoter and terminator fusions to make the linear constructs.
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Use LEEs as IVT as template. ASU has built the IVT templates.
Construction of initial set of ORFs in LEE expression constructs to be used as IVT
template
 Figure on slide 7 shows fully constructed IVT templates that can be used in
IVT reactions.
 Cheryl Selinsky generated data showing robust and normalized expression,
as much as possible. Cheryl tested coupled vs uncoupled reactions. The
uncoupled reactions gave robust product and more reliable product than the
coupled reactions. ASU has QC’d each step independently. The uncoupled
reactions give them more control over the pathway of reactions. In the high
throughput process, you want the higher yield and want the optimized
reactions for transcription and translation, independently. Coupled mixtures
don’t give the best optimization for both transcription and translation.
 Question: Joe- do you sacrifice throughput because you have to stop and
check? Answer: Kathy- it isn’t really a problem. We can robotically pull,
aliquot and QC the RNA template while other steps are proceeding. It takes
some time cost, but relative to synthesizing the final protein, knowing the
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steps where a problem arises, and not having as much control on the
template going into the reactions, the benefits outweighs the time cost.
Question: Joe- were you having more up/down results when it was coupled?
Answer: Kathy- yes, and it was harder to troubleshoot as you didn’t know the
step at which the problem arose. In the old literature, the coupled reactions
were designed for ease. The coupled may work well for a small number of
samples, but doesn’t work well for high throughput. With high throughput,
setting up two reactions is not as hard as assuring that repeats are not
necessary.
Question: Joe- have you had problems with RNA stability? Answer: KathyNo, because you have more of a problem in the coupled. It is hard to setup a
translation mixture that is completely RNase free. When you use separate
reactions, you can better optimize the reaction mixtures for the separate
DNA and RNA templates needed for transcription and translation,
respectively.
Question: Joe- the ORFs are they truncated to the optimum size as the photo
shows a variety of sizes? Answer: Kathy- this example set was performed
with varying length ORFs for these full length genes but for the high
throughput, all the ORFs for the library are 500-700 with an average around
600bp. So the size will be more consistent. This example set of genes was
chosen to work in pilot assays at UNM.
Cheryl shared this excellent data, with very good products off the in vitro
templates that ASU designed. ASU was able to evaluate the coupled vs
uncoupled transcription and translation reactions, and select the uncoupled
as the optimal approach.
Question: Rick- what is the yield anticipated? Answer: Kathy- for the first
batch of 25 ug/protein made, ASU will need multiple reactions but for the
high throughput a single reaction is likely to give 10-20ug/protein made. For
10ug output protein, they can use one reaction. They don’t want to have
multiple reaction wells with some and single reaction wells with other protein
products- it is more complex to set up robotically.
D. Milestone 32: Oligos selected for microarray production; Oligos list refined, 70mer
oligos procured, GDP oligos defined, Based on SCHU S4 sequence.
1. 70mer oligonucleotides ordered and received
2. Stocks and master printing plates made
3. GDPs received
E. Milestone 33: Printing and Testing and GDP confirmed
1. Have performed test spotting on 18 PLL and on 10 UltraGAPs slides, with 48 pin
configuration for printing. They have full genome prints completed on these
slides, with 5 replicates per probe printed. Spot sizes are 150 to 200microns.
2. Slide 10: Red reflect image of full genome print, Each gene probe in quintuplet
3. Slide 11: QC testing of Francisella RNA by Bioanalyzer- see beautiful integrity of
16 and 23S ribosomal RNAs on the Agilent Bioanalyzer result. The RNA integrity
is beautiful. The RNA has been used for cDNA synthesis and they will be
labeling it and performing hybridizations soon. Mitch used random primers in the
reverse transcription to make the cDNA from the purified bacterial RNA. This will
be used to QC the slides and later will do dilution series of the RNA, with the
GDP in the RT.
Goals for November
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Project 4, Proteinomic Approach
 Produce by IVT up to 8 designed fragments and send 20-25 micrograms
to Dr Lyons
Project 5, Transcriptome Approach
 Perform test hybridizations on poly-L-Lysine vs Ultragaps slides
 Perform amplifications (aka cDNA reverse transcription) with GDPs
 Dose response for limits of detection
 Coordinate with UNM to begin acquire samples Milestone 34- test tissue
sampling. UNM can begin gathering samples now.
 Action: Mitch- tell UNM what RNA samples are needed from test
tissues, perhaps from defined dose exposed lungs etc. UNM
could have the samples within a month for ASU; UNM must
assure that the tissue RNAs are decontaminated by culture
before the RNAs can be sent. Give UNM a list of RNAs desired.
 Question: Should we obtain TIGR microarray slides for comparison
studies? See discussion below.
TIGR Slides Discussion:
 Mitch has the application for obtaining the 150 slides. Mitch has
Kimberly Simple as the contact at NIH for submitting the application.
 The probes on the slides represent LVS and SCHU S4 genomes. Rick
thinks they looked for overlaps. The one library represents both
genomes. Rick said UNM has a combined vaccinia and cowpox, which
is similar to the LVS and SCHU S4.
 Mitch could do an alignment with LVS and SCHU S4 to see where ASU
might add probes to cover LVS genome too.
 Action: get the sequences of the probes from TIGR so Mitch can
determine whether the GDP primers from ASU will give rise to products
that hybridize to the probes on the TIGR arrays. Mitch needs to provide
a justification for needing the probe sequences.
 Joe: the TIGR slide surfaces may be different from PLL and UltraGAPS.
Mitch thinks it is a Corning slide, but possibly a different type. If ASU is
working out hybridization conditions, then the slide type is important.
TIGR is designed for distribution and the UNM TVDC is not budgeted for
this.
 Joe: says they ship them in batches of 25. Recommends put in one
request for slides and separate request for sequences. Don’t couple the
requests to TIGR to keep the slide procurement go faster. Maria
Giovanni is project officer and Joe can get her in touch with Mitch needs
assistance to get the sequences of the probes on the TIGR slides.
Other Agenda Items
A. Consortium purchasing?
 ASU: luminex beads, animals, and immune elisa reagents. Reagents are used for more
than TVDC project so split payment over TVDC and other funding sources.
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Marlene: Ross said all purchasing has nothing to do with the TVDC- UNM and DVC
charge back to the contract and NIAID has no involvement in the quotes.
 DVC , ASU, UNM- invitro translation enzymes, luminex beads, monoclonal antibodies for
cell identification, miltenyi beads for blood purifications after vaccinations.
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Multiple institutions: get a quote from a vendor using the combined volumes and try to
get lower pricing.
 Action: Mitch- have list of expensive reagents and vendors for Barbara on 10/26.
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B. Milestone Completion Reports: Action: Barbara will call Mitch at 9 am ASU time and 10 am
MT on Thursday 10/26.
C. Action: Kathy prepare the proteomics reagents presentation for Woods Hole.
Next ASU Tech Call
Tuesday, November 21, 2006. 11am-12pm PT, 12pm-1pm MT, and 2-3pm EST.
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