UNM TVDC: ASU - UNM Tech Call Minutes 11/21/06
Prepared 11/29/06: Barbara Griffith
Edited 12/5/06: Kathy Sykes and Mitch Magee
Sent to NIAID : 12/5/2006
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 from previous meetings:
1.Mitch will send Barbara an example of probe QC verifications for the thermal cyclers
Action Items from 11/21/06 meeting:
1. Kathy/Cheryl: ship peptides to UNM with list with locations; ship on a Monday 11/27 next week
(completed 11/27/06)
2. Barbara- arrange call on Thursday 11/30 (call scheduled for 12/4/06 Monday)
3. Terry/ UNM needs to isolate RNA from mouse organs that are infected; need to discuss further
with Mitch
4. Terry/ UNM team: needs to isolate additional purified RNA and DNA from LVS and SCHU S4
and ship to ASU
5. Mitch: obtain LVS and SCHU S4 slides from TIGR and LVS probe sequences from TIGR
Discussion of the Progress of Milestones
A. Active/Inactive Milestones:
a. Milestones – Sykes
i. Completed 25
ii. Active – 26
iii. Inactive – 28-30
b. Milestones – Johnston
c. Completed 32
d. Active – 33-34
e. Inactive – 35-38
B. Milestone 25: Design protein-fragment library based on SCHU S4 sequence
a. Computer program for oligo prediction is complete
b. 500 peptides to be arrayed and used in T cell assay workup have been received
at ASU and were sent to UNM on 11/27/06; Kathy thought the re arraying would
be better performed at UNM for peptide integrity. The disadvantage is that none
of the peptides would be banked at ASU. Rick and Kathy agreed that UNM will
test each peptide in a T cell assay separately first and then begin mixing the
peptides to increase complexity of the pool and to determine the impact of the
complexity on the ability to detect a response to a known reactive peptide. So
once UNM has identified some individual peptides that stimulate, then UNM will
start mixing. Currently, the 500 peptides are in 96 well plates and lyophilized.
c. Action: ASU ship peptides to UNM with list with locations; ship on a Monday
11/27 next week (completed 11/27/06); UNM develop testing protocol.;
d. Action: Barbara- arrange call on Thursday 11/30 (call scheduled for 12/4/06
Monday)
C. Milestone 26: ORF library Production
a. Design HTP ORF synthesis algorithms and SOPs, Test production, Confirm gene
expression, and Generate optimized ORF library
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b. Overview of Method for Expression Construct Assembly
i. Synthetically build promoter and terminator elements.
ii. Synthetically build or amplify SCHU4 open reading frames (ORFs) as per
new SynBuild software.
iii. Construct LEEs using optimized promoter, ORF, and terminator
elements.
iv. Use LEEs as in vitro templates for full scale protein production.
v. LEE: linear expression element made with cutting/pasting with no
bacterial involvement; cleaner than bacterial produced circular templates
c. Construction of IVT templates: LEE and plasmid versions, with and without
ubiquitin fusion, with and without biotinylation.
d. LEE templates are being further tested in coupled in vitro trxn/transl versus
uncoupled in vitro trxn/transl.
i. Cheryl has tested Calmodulin, GFP, and beta globin templates for a total
of 5 now. Previously, results were described for 2 others.
ii. ASU prefers the highest quality IVT which is Invitrogen coupled reaction
and is pleased with the templates. The LEE is working as well as the
plasmid templates. ASU will follow through on parallel with the feeder
system
iii. Question: Is the feeder system commercial? Kathy: It is not but we are
pretty communicative with Invitrogen. They are using it.
e. In vitro production of protein from linear DNA and RNA templates
i. Will use highest quality invitrogen coupled reaction = CURRENT
DECISION
f. Linear templates for IVT
i. If uncoupled reactions are feasible, they will be preferable since this
protocol will afford closer quality control, and therefore more consistent
output.
ii. Preliminary data suggest uncoupled reactions will be possible but
coupled may be preferable for high throughput and quality of the
commercial reagents available.
g. Discussion:
i. Joe: what is the difference between the terms “design and form” in this
context?
ii. Design: construct design with various tags, ubiquitin, ORF itself
iii. Form: RNA or DNA as template which means uncoupled versus coupled,
respectively. The form will be linear.
iv. Transcription: A linear template creates a “run on” reaction and thereby
we get more products than with circular plasmid. We are getting twice as
much RNA product in reactions that use a LEE as template relative to
the amount of RNA generated in reactions that use a plasmid template.
v. Translation: The coupled lysates – which are simpler to use-have been
industry-optimized, while the uncoupled ones have not. Therefore the
lower RNA amounts generated in a coupled reaction are compensated
for by higher translation efficiency. Hence, similar amounts of final
protein product are generated in either system.
vi. Joe: uncoupled could limit the throughput, but if product is better, then
use uncoupled. Coupled will have some misses, but overall, it is
probably not that different from uncoupled
vii. Kathy: linear run on template is better than circular template for in vitro
transcription. However, Promega makes the only commercially
available uncoupled lysates, and these translation lysates are not
optimized—whereas the coupled reactions are . Roche and Invitrogen
only sell coupled lysate systems and the Invitrogen is high quality.
viii. Joe: Coupled may give a better yield if translation is occurring
simultaneously with transcription.
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ix. Kathy: Higher quality invitrogen coupled reactions appear to be the best
decision with all considerations. In parallel, a lysate feeder system is
being investigated.
x. Alex is taking over the Cheryl’s activities on the in vitro translation
program.
D. Milestone 33
a. Discussed: Microarray substrate comparison photo
i. Corning UltraGAPS vs PLL on powerpoint slide 9. The goal is to
compare the two substrates for printing and hybridizing slides ( Ultragaps
vs PLL).
ii. On each slide, there will be >1800 genes, rRNA probes as controls; this
gives you an array of approximately 10,000 spots with each spot
replicated 5 times.
iii. The slide shows cDNA derived from a SCHU S4 RNA labeled with Alexa
555 and then hybridized to PLL and Ultragaps printed slides.
iv. On the PLL hybridized slide, the feature size is about 50% bigger
between 150-175 microns whereas on ultragap the feature size is about
100-120 micron.
v. Powerpoint slide 10 shows the average median values and this is not
impacted by the feature size. The PLL images are very nice with almost
twice the average intensity, low background, good signal to noise ratios
of 40-75. These results indicate that the PLL has advantages over the
UltraGAPS. Poly-L-Lysine slides have more dynamic range (~ 2 fold)
vi. The test arrays are going well and ASU is ready to start with PLL and
move on to the Genome directed primers for the cDNA synthesis.
b. Bioinformatic comparison of LVS to SCHU S4 coding regions
i. 135 LVS genes would not be detected with the current SCHU S4 probe
set
ii. 216 would have at least one nucleotide mismatched with the current
probed set. Those that differ by a single nucleotide out of the 70mer
would not be detected. Stephen thinks that with 3-5 mismatches, would
see difference in intensity.
iii. 170 >3 mismatched nucleotides
iv. 157 > 5 mismatched nucleotides
v. Plenty of room on the slide for more probes and Mitch estimates that it
would Cost $2000-3000 more to add LVS unique sequences to the
microarray slides.
vi. Joe: Will we be testing RNAs from animals infected with LVS or will we
be focusing on SCHU S4 infected animals?
vii. Mitch: has LVS pure RNA and will be able to test empirically, how well
the SCHU S4 probes hybridize to cDNA made from LVS RNA.
viii. Rick: mostly, the experiments will be based on SCHU S4 expression
over time in animals, though not very expensive to add LVS if needed.
ix. Mitch: could enhance the array in the future very well and easily in the
future; could use an extra plate to print LVS specific probes on the slides
with minimal disruption.
x. Question: Has ASU registered with TIGR to get their arrays?
1. This is in process, but not finalized.
xi. Mitch: We are on the process of doing that. We are going to do the
comparison between TIGR arrays and our arrays.
xii. TIGR arrays do have all the LVS specific probes. Perhaps ASU could
get the sequences of the LVS specific probes and ASU could compare
results off TIGR and ASU probes.
xiii. ASU can do the bioinformatics and get the design done. But again Joe’s
point is well taken. The TIGR slides are free and if for any reason we are
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just asking for LVS activity without genome directed primers the TIGR
slides will be available too.
xiv. Mitch: 183 GDP primers have arrived at ASU and were designed with
software from ASU to fit toward 5’ end of the transcript to amplify around
the probe region. This is similar in number to Anthrax(around 200 GDP) .
For other organisms, ASU uses closer to 45 GDPs.
xv. Action: Terry/ UNM needs to isolate RNA from mouse organs that are
infected; need to discuss further with Mitch
xvi. Action: ASU needs additional purified RNA from LVS and SCHU S4.
Labeling at 10ug /slide so will take as much RNA as we can give him.
Also needs more DNA for 3 color normalization. UNM will provide.
xvii. Barbara: Mitch do you need purified RNA both from LVS and SCHU 4?
About how much do you want?
xviii. Mitch: More is better. We are still labeling 10ug/slide and I have been
focusing on one color experiment and Stephen wants me to do tricolor
experiment with the genomic DNA normalization. So I have to push
those through.
xix. Do you need some DNA as well?
xx. Mitch: We have a little bit but may be we need more. We will be testing
the tricolor labeling system and the genome directed primers in the next
month.
E. ASU-TVDC December Action Items
a. Project 4, Proteomic Approach
i. Finalize selection of template arrangement and reagents
ii. Prepare to generate test protein-fragments and send 20-25 micrograms
to Dr. Lyons lab
b. Project 5, Transcriptome Approach
i. Finalize test hybridizations on poly-L-Lysine vs Ultragaps slides
ii. Perform comparisons of LVS to SCHU S4 RNA
iii. Perform amplifications with GDPs
iv. Dose response for limits of detection
v. Coordinate with UNM to begin acquire samples Milestone 34
vi. Question: Synthesize additional oligos for LVS?- will perform the design
but will hold off at this time to buy the oligos for LVS specific genes.
Next ASU Tech Call:
ASU Tech call: December 20, 2006 Wednesday noon-1pm MT (2-3pm ET)
ASU Tech call: January 30, 2007 Tuesday noon-1pm MT (2-3pm ET)
Personnel
Alex and Cheryl: Shifting roles. Alex is very experienced at high throughput molecular systems.
Kathy has moved Cheryl off this particular program and Alex is taking it on.
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