UNM TVDC: ASU - UNM Tech Call Minutes 03/27/07
Prepared 3/31/07: Barbara Griffith
Sent to ASU: 3/31/2007
Edited by: Kathy (4-3-07), Mitch, Stephen (4/4/2007)
Sent to NIAID: 4/4/2007
Present: Kathy Sykes, Mitch Magee, Barbara Griffith, Rick Lyons, Joe Breen, Stephen Johnston,
Ann Sutton
Absent: Kristin DeBord, Freyja Lynn, Vicki Pierson, Alex Borovkov, Marlene Hammer,
Action Item from 2/27/07 meetings:
1. Kathy will estimate the additional cost if a feeding system is used to produce the proteins
Action Items from 3/27/07 meeting:
1. Stephen: will contact the Invitrogen Sales rep and convince that bulk ivt feed system is
the best option, rather than individual columns
2. Kathy: May be able to send 6 purified and unpurified polypeptides to UNM within a
month.( end of April/early May)
3. Mitch and Elizabeth will have GDP priming of bacterial RNA done and the first analysis
completed before the site visit on 4/10
4. Barbara- look at the subcontract and prime contract to determine time to review, before
publication (See Article 29 of the ASU subcontract, below). 90 days is listed in the
subcontract (completed 3/31/07)
A. SLIDE 1: TVDC Progress Report for ASU 3/27/07
a. MS 25 and 32 completed; MS 26,28, 33,34 active
B. SLIDE 2: Milestone #26 Flow Chart: Prepare a high throughput protein production
system
a. Main concentration is on the purification protocols this past month
C. SLIDE 3: Previous Status
D. SLIDE 4: Current Questions
a. Significant question because enzyme adds costs and more DNA template does
not add cost
E. SLIDE 5: FTU polypeptide in feed system
a. S35 met labeled polypeptides, same volume loaded per lane,
F. SLIDE 6:Effects of feed, extra template and RNA pol on FTU polypeptide yields
a. Adding more template has a much greater effect than adding more enzyme
(Polymerase)
G. SLIDE 7: Conclusion
a. Template addition,
b. Joe: what are the counts? Is the expected product the lower molecular weight
band?
c. Kathy: precipitable counts, only new translation products get radiolabeled. The
prominent band of 25 Kd is the expected band, not the weaker lower molecular
weight band.
H. SLIDE 8: Previous Status
I. SLIDE 9: Current Question:
a. Do we have enough material to try a purification? We are switching the BAP site,
so don’t have biotinylated material. However can test purification vis His tag
J. SLIDE 10: ExpressionTemplate
a. His tag is at C terminus and biotinylation is at the N terminus
1
K. SLIDE 11: IVT purification
a. Eluted band is faint but is correct size. Only seeing full length products. Gel was
loaded by constant 4000 cpm per lane. Lane 2 is smear of bands. Lanes 1&4
have 4000 cpm each in the load.
L. SLIDE 12:Raw TCA-precipitable counts
a. 36% left on beads as Hetal didn’t do a second elution
b. Next experiment will include a second elution and hope will get better recovery at
approximately 50%
c. Stephen: realistic recovery is around 50% in the elution fraction
d. Rick: hard to get off the beads?
e. Stephen: nickel beads have only certain capacity, binding/release
f. Kathy: 10-20% of the counts could be partial products
M. SLIDE 13:GFP purification via C-terminal His tag
a. Stephen: if get a little more recovery at the expense of higher background, would
this be a problem?
b. Rick: do we need the purification, in the end?
c. Kathy: Cheryl tried skipping the purification of an ivt lysate for use in a T cell
assay. She didn’t see nonspecific stimulation with the full ivt lysates in an
ELISpot assay.
d. Rick: UNM can run assays in parallel with purified ivt product vs. unpurified ivt
product; Will do the initial set of 6 polypeptides this way.
e. Joe: how fast can the first 6 polypeptides be ready?
f. Rick: could do now with currently vaccinated primates. UNM can do the cellular
stimulation assays within 1-6 weeks of having the polypeptides
N. SLIDE 14:Conclusions
O. SLIDE 15:Purification work-up Plans
a. Rick: will do proliferation and gamma interferon Elispot assay as well
b. Kathy: use 6 initial ORFs to compare and contrast, purification vs. non purified,
will use low level S35 in all the samples to facilitate tracking/QC along the
synthesis and purification process
c. Stephen: If can make polypeptides from ORFs that stimulate without purification,
then can save effort.
d. Kathy: will provide Rick enough polypeptide of purified and unpurified to compare
in parallel, before deciding whether the purification is needed for the T cell
assays
e. Kathy: the feed system adds an extra cost and ASU has estimated the cost.
Feed whole 96 well plates in a dialysis tank of feed with substrate etc rather than
use individual columns to feed. In bulk, the rep doesn’t want to support due to
Roche liability problems. Vendor wants the individual little columns to be used.
Kathy prefers high volume feed system.
f. Rick: let empiric testing guide the decision
g. Action- Stephen: will contact the Invitrogen Sales rep and convince that bulk feed
system is the best option
h.
P. SLIDE 16: 6 FTU Pilot preps
a. 6 ivts in triplicate, use small amount of S35, one set with Bir ligase, measure total
precipitable counts in product, run on gels and transfer data to database,
compare unpurified vs. purified on Nickel and on streptavidin
b. Stephen: is S35 a problem for transport?
c. Rick: UNM can receive S35 and S35 doesn’t interfere with cellular assays at
UNM
d. Kathy: dilute S35 Met with cold Met, and still trace polypeptide with counts; cpm
will be below the federal regulations for S35 transport
Q. SLIDE 17: MILESTONE 28 Flow Chart: Build SCHU S4 Proteome
R. SLIDE 18: Previous Status
a. Good: no problems amplifying and have codons that work
2
b. Kathy: can they make the ORFs
S. SLIDE 19: Question
T. SLIDE 20: 1st set of FTU ORFs selected for PCR production
a. Example one gel showing first round or first pass of ORFs produced by PCR.
The PCR is generally optimized but not specifically optimized for all 72. 68/72
look good with the general optimization.
b. Andre repeated the 4 that had failed and they gave PCR products successfully
on the 2nd pass.
c. Kathy: expects most failures from first round will be successful on the 2 nd round
d. Kathy: Do large batch of ORFs, then go back and repeat all the prior failures in
the 2nd round pass
e. Stephen: If 5% of the successfully produced ORFs are likely to work in later
cellular assays, and ASU gets 96% successful production in the first round, it
may not be worth the 2nd round pass for the few first round failures.
U. SLIDE 21: Conclusions
V. SLIDE 22: Work plan for upcoming month
a. Kathy: may be able to get these polypeptides to UNM within a month or so.
b. Rick: UNM has plenty mass of the original peptides
c. Kathy: will have the first 6 polypeptides to UNM by end of April
d. Joe: the set described in slide 16?
e. Kathy: yes
W. SLIDE 23: MILESTONE 32 Flow Chart: Oligo List refined, 70mer oligos procured,
GDP oligo defined. Will be based on SCHU S4 genome
X. SLIDE 24: Microarray Slide Comparisons
a. MTA needed to get the TIGR slides to ASU
b. Stephen: The TIGR slides are getting treated as if biosafety hazard by ASU, but
these are just slides and pose no biosafety hazard.
Y. SLIDE 25: MILESTONE 33 Flow Chart- Printing and testing GDP confirmed
a. Stephen: GDP are “genome directed primers”. Small amount of whole infected
organ RNA is actually the bacterial RNA so GDP preferentially amplify the
bacterial RNA
Z. SLIDE 26: Previous Status
AA. SLIDE 27: GDP Testing
a. Mitch: planned to complete last week. Linear amplification of RNA. Will amplify
600 to 1000 fold and want to compare to unamplified RNA.
b. Stephen: There is no poly A tail on bacterial RNA so use GDP to amplify the
bacterial RNA specifically in the background of the host organ RNA; can’t use
random primers for the amplification as random primers would amplify the host
organ RNA as well as the bacterial RNA.
BB. SLIDE 28: MILESTONE 34 Flow Chart- Pilot studies of optimization of RNA isolation
and hybridization conditions
a. ASU has received the infected lung RNA from UNM
CC. SLIDE 29: Problem Identification
a. MTA has caused the delay for the TIGR slides
b. Equipment failure delayed the ability to due amplifications of LAR
c. Stephen: Z scores of all genes need to remain constant when the bacterial RNA
is diluted in the mouse organ RNA, and then amplified with the GDP. Mitch tried
a different amplification and won’t use that method again
d. Stephen: do extraction, GDP priming, and then the amplification of the GDP
primed material; can get 3000 fold amplification of the bacterial RNA, with Z
scores essentially the same for the genes. ASU team has utilized the GDP
approach successfully with 3 other bacteria.
DD. SLIDE 30: Work plan for upcoming month:
a. Mitch: down to 1000 fold diluted bacterial RNA, then do bacterial RNA diluted into
control mouse lung RNA, then will follow with infected mouse lung RNA
b. Stephen: once GDP’s are validated, then won’t need a large quantity of infected
3
mouse lung RNA for future assays
Mitch: compare TIGR vs. PLL and Corning slides; compare SCHU S4 RNA by
GDP amplification
d. Stephen: Mitch and Elizabeth will have all done and first analysis before the site
visit.
e. ASU has the rat and mouse infected RNAs from UNM and day zero as well as a
control
f. Stephen: ASU is making 200,000 peptides per immunosignaturing slide, as an
FYI.
c.
Additional Discussion: National Meetings and Publications
 Stephen will be a keynote speaker at the Proteomics conference in May
 UNM and NIAID need to review slides before presentations and papers before
publication, if the data was generated from the TVDC.
 Joe: typically 45 days in advance is sufficient
 Action: Barbara- look at the subcontract and prime contract to determine time to
review, before publication (See Article 29 of the ASU subcontract, below). 90 days is
listed in the subcontract (completed 3/31/07)
 Joe is attending proteomics meeting at the end of May, as are Stephen and Mitch
Next ASU Tech Call:
ASU Tech call: May 22, 2007 Tuesday noon-1pm MT (2-3pm ET)
Semi- Annual Report due 4/7/07
Site visit: 4/10 at ASU; Joe cannot attend but Vicki and Marlene will attend, along with Rick and
Barbara
ARTICLE 29
COPYRIGHT AND PUBLICATIONS
SUB may copyright material developed in the course of this Agreement and to permit
others to do so. UNMHSC and the U.S. government shall have a royalty-free, nonexclusive, irrevocable license to reproduce, publish, or otherwise use, and authorize
others to use, any such copyrighted or copyrightable material. SUB shall place an
acknowledgment and disclaimer, as appropriate, of federal government support on any
publication written or published with funds from this Agreement and, if feasible, on any
publication reporting the results of or describing activities under this Agreement. The
acknowledgment shall be to the effect that “This project has been funded in whole or in
part with Federal funds from the National Institute of Allergies and Infectious Diseases,
National Institutes of Health, Department of Health and Human Services, under Contract
No. HHSN266200500040C.” SUB shall incorporate the requirements of this section in all
lower tier subaward agreements and grants.
SUB shall submit to UNMHSC an abstract for any proposed publication or presentation
ninety (90) days prior to publication to allow UNMHSC and Sponsor to review content of
the proposed publication or presentation and to secure protection for any confidential
information contained therein.
4