UNM TVDC: ASU - UNM Tech Call Minutes 12/20/06
Prepared 12/20-30/06: Barbara Griffith
Sent to ASU: 12/30/06
Edited: Kathy Sykes and Mitch Magee 1/9/2007
Sent to NIAID: 1/9/2007
Present: Kathy Sykes, Mitch Magee, Marlene Hammer, Mindy Tyson, Barbara Griffith, Rick
Lyons, Joe Breen, Vicki Pierson
Absent: Kristin DeBord, Freyja Lynn, Stephen Johnston
Action Items 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
Action Items from 11/21/06 meeting:
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 - 12/20/06- is in progress
5. Mitch: obtain LVS and SCHU S4 slides from TIGR and LVS probe sequences from TIGR12/20- is in progress
Action items from 12/20/06 meeting
1. Kathy will show data for other 7 proteins synthesized from the linear templates in next
technical report.
2. Barbara will send Mitch the chemical blocking protocol for PLL slides. (completed 1/8/07)
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
i. Completed 32
ii. Active – 33-34
iii. Inactive – 35-38
B. Milestone 25: Design protein-fragment library based on SCHU S4 sequence
a. Computer program has been written for ORF recoding and oligo prediction for
gene synthesis.
b. ORF designs are complete
c. Completion report will be sent to UNM around 1/15.
d. Aim addendum: design synthetic peptides for testing alongside the protein
fragments in the preliminary T cell assay workups.
i. Test peptides to be used in Tcell assay workup have been selected,
synthesized, and received by UNM.
ii. The 500 designed 20-mers have been sent from ASU to UNM as
lyophilized samples (~4 mg) in microtiter plates. These peptides
correspond to tularemia protein sequences that have been predicted to
be MHC Class I or Class II epitopes.
iii. UNM/ASU held conference call to plan use, testing, and storage of the
peptides
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iv. Note: Rick says some peptides individually stimulate and some don’t;
this is good news; splenic proliferation vs peptides using mouse cells .
20mers are working
v. Mitch: have immunized with cowpox genes for another project and using
the peptides of the same design and size in T cell assays. In these
experiments, ASU used Interferon Gamma induction -as measured by
ELISPOT, rather than cell proliferation as a readout for T cell activity .
C. Milestone 26: ORF library Production
a. Design HTP ORF building protocols
b. Confirm gene expression of ORFs in linear template constructs
c. SynBuild software and design algorithms are completed. The software in
conjunction with the molecular biology advances, allowed high fidelity,
inexpensive and fast synthetic gene building.
d. The disclosure is for the gene recoding design concept and the lab protocols
which allowed the chip based synthetic gene synthesis. The disclosure is an
internal document that will used by ASU to evaluate whether they should submit
a provisional patent application. Internal disclosure is the first step before a
patent application. AZTe is a team of lawyers and intellectual property
specialists for ASU. SynBuild was developed for the TVDC and an FTU grant
collaboration with Rick; the project supported by TVDC, the FTU P01, and
internal funds.
e. Vicki: How will disclosure impact to meet deliverables if ASU decides to pursue a
patent? NIAID’s aim is to make the deliverables publicly available and not
restrict access. Is NIAID’s aim and ASU's potential patent in conflict?
f. Kathy: All the generated reagents would be made available. Software would be
copyrighted and then made publicly available at CIM, the Center’s Website at
ASU. The patent could be on the concepts of the gene design and the protocols
to synthesize the genes using microfluidic chip based synthesis of oligos. None
of this is compromises any tularemia reagents, but rather enhances them.
Deliverables on the contract would not be impeded. The software would be
publicly available and downloadable from the CIM website and the reagents
would be available to anyone. The point of the disclosure is that the intellectual
property is more valuable than just the actual oligos synthesized . ASU will be
delivering blocks, pieces of genes that will be easily amplifiable from one
common set of PCR products so it is a high quality renewable resource which will
be available for anyone. Without the technology development, ASU could only
deliver the thousands of oligos, which the end user would have to assemble in a
highly technical protocol. With the technology development, the end user can
use the units or blocks more easily to generate genes—with very little technical
skill. The user could utilize the software and block technology to make genes
from any pathogen in the future. Anyone could use the software for future.
Concept of using chip based synthesis is the pre- patent disclosure.
g. Generate test protein fragments to give to UNM
h. Build complete ORF library
i. Evaluation of Linear Templates for in vitro ORF expression
1. Showed the gel of the results of the protein synthesis using a
variety of linear templates.
a. GFPexpressed with plasmid template,
b. calmodulin expressed with plasmid template,
c. calmodulin expressed with covalent LEE template,
d. calmodulin expressed with linked LEE template,
e. calmodulin expressed with linked LEE template, short
promoter,
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f.
2.
3.
4.
5.
6.
calmodulin expressed with linked LEE template, short
promoter and biotin attachment site,
g. calmodulin expressed with plasmid template, short
promoter,
h. calmodulin expressed plasmid template, short promoter
and biotin attachment site.
From the most simple to the most complex/flexible template, all
linear templates gave equally sufficient amounts of protein. So
can add biotin or shorten promoter successfully. Adding these
other elements was more risky potentially for protein quantity or
purity, but they caused no problems and will be advantageous.
So elements may have helped folding but hurt purification or may
have helped purification at the cost of quantity of protein
synthesized. Adding the desired elements was risky from this
perspective, but beneficial if the elements worked as desired.
During HTP IVT reaction the Bir enzyme adds the biotin directly
while the protein is being synthesized simultaneously. The biotin
can be cleaved off the protein product before use, if desired.
Joe Question: has done calmodulin which is 400 aa and FTU’s
as well – 7 other proteins have been synthesized as well and all
have been designed within the same size range. Action: Kathy
will show data for other 7 proteins synthesized from the linear
templates in next technical report.
Have done test IVT reactions in presence of Bir. These are now
being repeated so as to prepare material to evaluate the biotin
purifications. Biotin-based purification protocols are currently
being tested.
Conclusions:
a. These results show that similar amounts of product are
generated under all conditions tested.
b. We conclude that our recombinant LEE attachment
protocol works, that we can include a biotin binding site
to use for high efficiency purification, and a streamlined
T7 promoter can be used.
c. As confirmation, these results were reproduced in an
independently conducted confirmatory experiment.
D. Milestone 33
a. Microarray substrate comparison repeat: PLL vs Corning Ultra Gaps
i. Arrays printed on PLL-coated slides perform comparably to Corning
Ultragaps using median normalized signal intensities
ii. PLL and ultragaps have comparable signal to noise ratios
iii. PLL and ultragaps have comparable background signal intensities
1. PLL spot sizes are ~50% larger than the Corning ultragaps
slides
2. So visually, the differences appear greater than when you
analyze the median intensity value across the slide. PLL is a
little greater median intensity SLIDE 10, than Corning Ultra
GAPS
iv. Planning now to use PLL; no big advantage of Corning UltraGAPs
relative to PLL made in house.
v. Rick: UNM found this result in past slide comparisons and uses the PLL
vi. Mitch: their team makes better PLL slides than the commercially
available other slides. Mitch has studied the slides for peptide arrays
also. The substrate slide comparisons are now done.
vii. Action: Terry is working on more SCHU S4 and LVS RNA for Mitch.
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viii. Within next month, will amplify RNA with GDP and will compare ASU
slide comparison with TIGR slides.
ix. Barbara: recommends post processing within a couple of days after
printing and Action: Barbara will send chemical blocking protocol to
Mitch. UNM found that the blocked, printed PLL slides were stable for a
year after printing. The chemicals include succinic anhydride treatment
and then washes in ethanol.
x. Mitch: printing and using after storage at room temp; no blocking in the
ASU current protocol. ASU hasn’t done long term stability studies on the
printed slides.
xi. Barbara: how do you prevent non-specific DNA hybridization to the rest
of the coated slide, after you print?
xii. Mitch: the background is very low, less than 500 without blocking
xiii. Rick: maybe works due to simplicity of bacterial genome.
xiv. Rick: what is the data comparison on slide 10?
xv. Mitch: these are the normalized data intensities across the whole slide.
To compare the two slides, we examine the median overall data intensity
and standard error across each whole slide.
xvi. Rick: would have expected the difference between PLL and Ultragaps to
be much more different. Mitch: reminded that the spot sizes are different
on the two slide types with PLL having 50% bigger spots. When you
examine the median intensity values after spot finding and data
processing, the PLL and the Corning Ultragaps are not as different as
your eyes expect based on the raw images.
xvii. Rick: Action: UNM Needs to send ASU RNAs from mice exposed to
different doses to determine the infection dosing needed to see a
difference on the microarrays based on lung and spleen expression;
Once Mitch knows his detection limits, then call tell UNM the infection
dosing range.
xviii. Rick: This approach is looking for unique expression patterns in early
and late phases of infection that suggest good vaccine candidates.
E. ASU-TVDC December Action Items
Project 4, Proteomic Approach
 Finalize selection of template arrangement and reagents
 Prepare to generate test protein-fragments and send 20-25 micrograms
to Dr. Lyons lab First 500 peptides: testing complexity that can be used in in vitro
stimulation
 The smaller number of protein-fragments will be used to confirm
optimization: ASU picked 8 from literature for the protein fragments
 Tul4 is one of the proteins
 Joe: how successful is this quantity of protein (20-25ug) from an IVT
system? Mitch- haven’t reached this 25 ug level yet, but are
approaching this level
 Rick: want this optimized with protein fragments before moving to
primates; need to know the limits of detection with the mass of protein
 Joe: want to see the data for high quantity IVT and how well the
purification works to pull desired protein from the IVT background. The
IVT are not exact. Rick: we are currently testing whether we can get the
high purity and high quantity needed. Then will move to the production
phase. The 20-25 ugs is desired but we need the data regarding the
actual quantity feasible.
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Joe: reaction engines reach therapeutic levels but takes energy,
reagents and time. Engineering the biotin in parallel will assist the
purfications greatly.
Rick: we need thousands of proteins so it needs to be efficient process.
Project 5, Transcriptome Approach
•
Perform comparisons of LVS to Schu S4 RNA
•
Compare TIGR arrays to ASU arrays.
•
Perform amplifications with GDPs
• Dose response for limits of detection
•
Coordinate with UNM to begin acquire samples Milestone 34
Next ASU Tech Call:
ASU Tech call: January 30, 2007 Tuesday noon-1pm MT (2-3pm ET)
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