Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 1 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
Signature Page
Author’s Signature:
_Kathryn Sykes____________________
Typed Name of Author
Acceptance by Subcontracting Institution:
_Kathryn Sykes ____________________
Typed Name of Subcontracting PI
Acceptance by the University of New Mexico:
C. Rick Lyons, MD. PhD ______________
Typed Name of Principal Investigator
Acceptance by NIAID:
_Freyja Lynn and Patrick Sanz_______
Typed Name of NIAID Project Officer
_Heidi Holley______________________
Typed Name NIAID Contract Officer
__1/6/2010__
Date Accepted
Page 1 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 2 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
Table of Contents
1 Milestone Summary ................................................................................................................ 2
2 Milestone Objectives ............................................................................................................... 2
3 Methods, Critical Reagents and SOPs .................................................................................... 3
4 Salient Original Data, Results, Interpretation, Quality Control ................................................. 4
4.1
Original Data and Results (Rationale, Tables/Figures with legends and location
annotations) .............................................................................................................. 4
4.2
Interpretation .............................................................................................................14
4.3
Quality Control ...........................................................................................................15
5 Deliverables Completed .........................................................................................................15
6 Appendices ............................................................................................................................17
6.1
Appendix 1: Original Data Tables and Figures ..........................................................17
6.2
Appendix 2: Quality Assessment of Milestone Completion and Report .....................21
6.3
Appendix 3: Additional Data/Figures not included in the Text of the Milestone
Completion Report (Section 4) .................................................................................25
1
Milestone Summary
In MS26, ASU developed the laboratory protocols required for implementation of milestone 28.
The objective of milestone 28 was to deliver the complete proteome of F. tularensis (FTU) as an
arrayed set of proteins of sufficient quantity and quality to productively serve as antigen in T cell
immune response screen. The overall goal is to utilize a newly developed proteomic-immunome
approach to identify subunit or polypeptide vaccine candidates for Francisella tularensis. In
addition to defining the high throughput protocols themselves, ASU also established the data
management and quality assurance/control tests needed to ensure the polypeptides’ optimal
performance in the T cell based immune screen. These goals have been met. ASU delivered 2,229
FTU synthetically produced and affinity purified polypeptides to the University of New Mexico for
use as antigens in high throughput ELISPOT assays. The polypeptides have been evaluated,
catalogued, and utlized in T cell experiments. Arraying of the polypeptides into 319 pools of seven
polypeptides per pool was performed to streamline the cellular screen; any positive pools will be
partitioned to identify individual immunogens to be tested as vaccine candidates.
2
Milestone Objectives
The objective of ASU Milestone 28 is to generate a Fransicella tularensis polypeptide library and
to ship the arrayed polypeptides in pools to UNM for testing in T cell assays. To support this
objective, ASU will build the SCHU S4 proteome, by building the ORF expression library
corresponding to the genome and then generating the complete protein-fragment library
corresponding to proteome. ASU will implement a high throughput system based on Milestone 26.
Page 2 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 3 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
A location map of the peptide matrices is a deliverable. The map can be found in
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU proteomic library\Gems info and will be
provided in an Excel format.
3
Methods, Critical Reagents and SOPs
3.1
Methods:
3.1.1
DNA templates used for building SCHU4 proteome were constructed by overlapping FTU
ORFs with T7 promoter containing a thioredoxin tag and T7 terminator containing a 6X
Histidine tag by PCR amplification. Such a method was chosen to overcome the difficulty of
cloning a large amount of FTU ORFs using traditional plasmid techniques. T7 promoter was
amplified from Novagen pET32b whereas T7 terminator was amplified from pIVEX2.3d.
3.1.2
For cell-free protein synthesis, NEB PURExpress In vitro protein synthesis kits were chosen
since they contain minimal amount of cellular E.coli proteins which have shown cross reactivity
in ELISpot assay using murine splenocytes. Dynal beads bound thioredoxin antibody were
added into the reaction mixture prior to the incubation for protein purification. Bead bound
proteins were separated from the reaction mixture, washed, and stored in PBS buffer.
3.2
Critical reagents:
3.2.1
3.2.2
3.2.3
3.2.4
Novagen pET32b for obtaining T7 promoter
pIVEX2.3d for T7 terminator
NEB PURExpress in vitro protein synthesis kit
Magnetic Dynabeads M280 Tosylactivated beads from Invitrogen and monoclonal antithioredoxin antibody from Gene Script Corporation
SOP Number1
ASU-Proteome
SOP#3
SOP Title
High throughput construction and purification of
polypeptides
1
Individual Standard Operating Procedures will be reviewed separately and accepted by
the Subcontracting PI and UNM PI
Page 3 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 4 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
4
4.1
Accepted Date:9/11/09
Salient Original Data, Results, Interpretation, Quality Control
Original Data and Results (Rationale, Tables/Figures with legends and location
annotations)
Sub-milestone 28.1: Build ORF expression library corresponding to genome
In this section, ASU carried out the first part of the master protocol (Proteome SOP #3
Production and purification of polypeptide libraries). Primers were designed according to the
algorithms developed in MS25 and purchased. Using F. tularensis genomic DNA, the 2,229
primer pairs were used to PCR-amplify all intended ORFs. These genes were linked to
promoter and terminator sequences in a two-step overlapping PCR protocol as developed in
MS26. These linear expression constructs (LEEs) comprised the FTU ORF library. All LEE
templates were quantitated by UV-spectrophotometry (Nanodrop 2000) and also fractionated
in agarose gels to visualize quantity and quality with ethidium bromide. These data were
converted into an e-gel analysis program developed in MS25. The results generated from a
portion of these expression templates is displayed on the e-gel in Figure 1.
Page 4 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 5 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
F- 1. E-gel representation of LEE constructs comprising FTU ORF expression library. The LEEs were
assembled by overlapping of T7 promoter and terminator by PCR amplification in 96-well plate format. One tenth of
the volume reaction of each PCR reaction (10μL) was mixed with 10μL molecular graded water then loaded on the
gel. Each gel represents 84 PCR samples from 96-well plate.
Data location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU proteomic library\E-gel\Long ORF 2
Sub-milestone 28.2: Generate a polypeptide library corresponding to the F.
tularensis proteome.
The validated LEE constructs were used as expression templates in the protocols
developed in MS26 for in vitro transcription and translation. Before scaling up to full
production, ASU ran a few pilot experiments on the reagents which were purchased in
bulk to assure consistency across all polypeptide synthesis. For example, ASU tested a
large batch of the reconstituted IVT reaction mix that had been selected in MS26 in order
to establish the quality of this specially ordered New England BioLabs (NEB) reagent. A
row of 12 templates were expressed with the NEB IVT pure system in the presence of
radiolabel. The IVT products were fractionated in SDS-PAGE and visualized by
autoradiography. The results in Figure 2 show that the templates and reagents were able
Page 5 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 6 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
to generate high quality polypeptide products. In particular, the synthetic radiolabeled
chains are not degraded, they appear as full length as estimated by the predicted
molecular weights, and yields are sufficient for use in the intended assays.
F-2. NEB IVT Pure system reagent expresses row A of FTU LEE amplification PCR plate 2. One microgram of
LEE template was used for IVT reaction in which 3.3μCi 35S methionine was added for visualization. The total
reaction volume is 50uL; 1/10 of this volume was loaded on the gel (Biorad Criterion XT Bis-Tris 4-12%). Gel was run
at 150V for 55 minutes, dried, and transferred to a storage phosphor screen to generate the image.
Data location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT 35S gels\F tularensis
proteomic library\NEB IVT kit testing 10-8-02 crop 2
In another pilot experiment, ASU evaluated the storage stability of the bead-bound polypeptide products.
This was done by expressing OVA (control) and an FTU (FTU721a) gene template in triplicate reactions.
One sample was stored at 4 degrees C for 5 days, one was frozen at -80 degrees C for 5 days and then
thawed, and another was not stored at all but was used on the same day. These three unpurified but beadbound samples of two IVT templates were fractionated and visualized. The results in Figure 3 show that
no loss of integrity occurs in the samples following the storage and freezing procedures, relative to the
product integrity immediately after synthesis. Also, analyses of the bead supernatant following storage
showed that there was no reduction in binding of the polypeptides to the beads following 4 degree C
storage or a freeze-thaw.
Page 6 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 7 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
F-3. Testing stability of bead-bound IVT polypeptide samples with storage. Ova and FTU LEE were in vitro
translated using NEB PURExpress kit and purified using anti-thioredoxin magnetic bead bound antibody. After the
reaction was completed, beads were washed and resuspended in 75µL with PBS; 25µL from this volume was used
for each storage test. For test 1, the beads were separated from supernatant immediately after the IVT reaction is
complete. For test 2, the beads in PBS were stored at 4oC for 5 days. Next, they were separated from supernatant.
For test 3, the beads in PBS were stored at -80oC for 5 days. After 5 days, they were thawed on ice and separated
from supernatant. Separately, beads and supernatant were mixed with SDS loading buffer containing 5% βmercaptoethanol, heated to 90°c for 5 minutes. Supernatant samples were loaded directly on the gels, whereas only
loading buffer mixed with the beads was loaded on Biorad Criterion XT Bis-tris 4-12% gel. Gel was dried and
transferred to storage phosphor screen to generate the image. Arrows indicate bands corresponding to molecular
weights of full length Ova and FTU721A products
Data location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT 35S gels\Testing bead
storage temperature 2
Before scaling up production, ASU tested a small set of purification reactions using an aliquot of
the batch of anti-trx (thioredoxin) antibody purchased for the full library. A new large batch of
beads and monoclonal (mAb) were prepared and tested in the IVT polypeptide purification
protocol defined in MS26. Row A of IVT templates from FTU LEE amplification of FTU Long
ORF 2/PCR plate 2 were chosen for the test. The results presented in Figure 4 demonstrate that
the Trx mAb bound beads act as a highly efficient ligand for the polypeptides. Quantitative
levels of product are attached to the beads (odd lanes), while only marginal amounts are detected
in the unbound supernatant fraction (even lanes).
Page 7 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 8 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
F- 4. Evaluation of bulk ordered anti-trx antibody in purification protocol. Templates from FTU Long ORF plate
5/PCR plate 2 row A were used. The odd numbered lanes were loaded with IVT proteins eluted from beads after
washing (antibody captured sample). The even lanes (shown without numbers in the figure to avoid over crowded
labeling in the figure) were loaded with 20% of the bead supernatant removed prior to washing (uncaptured sample).
Quantitative levels of product are attached to the beads (odd lanes), while only marginal amounts are detected in the
unbound supernatant fraction (even lanes).
Data location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT 35S gels\F tularensis
proteomic library\ Testing large bach of beads and Ab 10-23-08 crop
ASU conducted a set of pilot IVT reactions with all reagents prepared for library production and protein
purification using radiolabel. These pilot products were evaluated for appropriate molecular weight,
purity and mass before scale-up of the full library. Templates carrying ORFs generated by both PCR and
gene assembly were tested. These showed robust yields and no degradation, as displayed in Figure 5.
Page 8 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 9 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
F-5. Autoradiograph of polypeptides generated from the ORF templates. After the IVT reaction complete, beads
were separated from reaction mixture, washed in PBS, and resuspended with SDS loading buffer. Samples were
heated to 90oC for 5 minutes. Only loading buffer of each sample was loaded on the gel. After drying, gel was
transferred to storage phosphor screen for image.
Data location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT 35S gels\F tularensis
proteomic library\BAG\QC PCR plate 1 and 2
Furthermore, these same products were visualized by Coomassie staining to evaluate their level of purity.
The gel shown in Figure 6 displays all products eluted from the antibody-beads. The heavy and light
immunoglobulin chains are visible in all lanes as expected. The synthetically IVT produced polypeptide
chain is visualized as a prominent additional component. These results confirmed a high level of
purification was achieved.
Page 9 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 10 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
F-6. Coomassie blue stain of unlabeled FTU polypeptides from gene-built templates. After the IVT reaction was
complete, beads were separated from IVT reaction mixture, mixed with SDS loading buffer, and heated up to 90°C for
5 minutes. The loading buffer containing the de novo polypeptides and anti-thioredoxin antibody was loaded on
Biorad Criterion XT 4-12% gel. Gel was stained with Invitrogen Simply Blue. Gel was imaged with ChemiDoc™XRS.
Data location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT Coomassie gels\F
tularensis Library\BAG QC plate 1 and 2 11-25-08 crop
With these pilot experiments complete, the remaining plates of templates were used for polypeptide
production. The full library products were not radioactively labeled; however, randomly selected sets of
templates (12 from each 96-well plate) were run in duplicate to serve as QC samples. The duplicate
reactions included 35S-met radiolabel for visualization and quantification of products. An additional
positive control template, encoding green-fluorescence protein (GFP), was included as a non-FTU gene
sample
The mass yield of each QC sample was determined by two methods. First, the amount of incorporated
radioactive 35S-methionine (met) label precipitated with trichloroacetic acid (TCA) was calculated based
on specific activities and the known number of methionine in each polypeptide based on the manufacture
calculation formula as shown in ASU Proteome SOP#3 Second, samples were fractionated in SDSPAGE and gray-scale intensity was measured by densitometry. For this experiment the antibody-captured
IVT products (calmodulin) were eluted from the bead-antibody conjugate prior to fractionation. Sample
band intensities were compared to a titration of protein standards (GFP). The recombinantly produced
GFP had been quantitated by BCA (bicinchoninic acid) protein assay, and run on the same SDS page gel.
The Coomassie stained gel of purified recombinant GFP used as protein standards and IVT-generated,
TCA-quantitated calmodulin is shown in figure 7. Based on TCA assay, the total yield of lane 2 is 0.97ug
compared to 0.90ug from densitometry method. The difference between calculation of IVT protein yield
from the TCA assay and densitometry method is approximate 7%.
Page 10 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 11 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
F-7: Coomassie stained gel of IVT Calmodulin before (1) and after (2) purification compared to the protein
standards with shown amounts. An Invitrogen control vector carrying the Calmodulin gene was used as the test
template. As protein standards, recombinantly produced GFP protein purified by nickel affinity column
chromatography (0.1 to 1.0ug/lane) was used.
NOTE 1: The GFP protein was recombinantly produced and nickel bead purified to obtain mass quantities of protein
as a concentration standard. Thus the GFP protein preparation does not contain other protein bands.
NOTE 2: ASU had already repeatedly demonstrated that the linear and circular plasmid templates perform similarly.
It was easier and less expensive to use plasmid templates for the developmental phases and then after development,
ASU performed a final confirmation of the optimized protocol with a PCR product as template. A PCR-produced or
gene-built produced piece of DNA will not differ in structure
NOTE 3: For two reasons, the purified calmodulin polypeptide in lane 2 of figure 7 does not show the immunoglobulin
chains that were visible in the lanes of Figure 6 above. Reason 1: The calmodulin polypeptide in figure 7 was eluted
from the bead-antibody conjugate, whereas the FTU polypeptides in Figure 6 were retained onto beads. So, when
the FTU samples (bead, Ab, polypeptide) were run on a gel in figure 6, the heavy and light immunoglobulin chains
were present and stainable. Reason 2: The calmodulin protein is well known to be very efficiently translated in these
in vitro reactions. There is always more calmodulin polypeptide made than other samples by these IVT lysates. So,
this results in the low levels of lysate components visible in F-6 but not visible in the small proportion of calmodulin
sample fractionated in F-7 as shown in lane 2.
NOTE 4: In F-7, lane 2, the FTU polypeptide was eluted from the beads as follows. An equal volume of .2M NaOH is
added to the beads/polypeptide/antibody complex to make a final concentration of .1M NaOH. The base
disassociates the Trx-polypeptide from the anti-trx antibody, but the antibody remains attached to the beads. The
beads/antibody complex is separated by the magnets and the supernatant containing the polypeptide is assayed on
the gel in Figure 7.
Data location: R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT Coomassie gels\ gfp
conc curve gray scale
Page 11 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 12 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
The autoradiographs of samples used for QC are represented in Figure 8. In total there were 8
QC plates: BAG (gene built), Short 1 and 2, Long 1-5 “Short 1” or “Short 2” plates contains ORFs which
are 600 nucleotide base pairs or less, whereas “Long 1-5” plates contains ORFs which are greater than
1200 nucleotide base pairs. “BAG refers to ORFs we expected to need to build. However, ASU was able
to optimize the PCR reactions sufficiently so as to amplify them instead. These SDS gels and
autoradiograph indicate that 96% of the DNA templates were translated into polypeptides with high yield
and integrity. In summary, ASU successfully purified microgram quantities of 2, 229 polypeptides
corresponding to the F.tularensis SCHU4 proteome. The average amount of proteins bound on beads was
15.8 ug per 25ul IVT reaction, The 15.8 ug/rxn yield was calculated based on the yields from eight 384
well plates; yields ranged from 7.7 to 29ug/rxn.
Fragment
Name
FTU0029C
FTU0080A
FTU0083A
FTU0090A
FTU0094A
FTU0101B
FTU0117A
FTU0136B
FTU0137A
FTU0146A
FTU0162A
FTU0184A
well
Loc
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
MW
39048.4
39441.9
29103.3
28945.2
41663.8
33037.7
35511.5
40915.4
35485.5
20488.0
31581.3
28942.3
#Ms
6
6
6
3
6
6
5
5
12
5
3
6
CPM
10420
26247
25430
13657
16831
29659
23337
21014
42466
29724
18452
23362
Total
count
104200
262470
254300
136570
168310
296590
233370
210140
424660
297240
184520
233620
Specific
activity
13.9
34.9
33.8
18.2
22.4
39.5
31.1
28.0
56.5
39.6
24.6
31.1
TCA
ppt
3638
10139
16777
2608
5212
9028
7718
8421
21237
9984
5231
8153
pmoles of
Met
incorporated
2546.6
2678.0
2736.9
1434.7
2326.5
2286.9
2484.7
3010.7
3757.2
2523.5
2129.9
2621.9
pmoles
of
protein
424.4
446.3
456.2
478.2
387.8
381.2
496.9
602.1
313.1
504.7
710.0
437.0
Yield
of
protein
16.6
17.6
13.3
13.8
16.2
12.6
17.6
24.6
11.1
10.3
22.4
12.6
T-1. FTU IVT protein yield calculation is based on number of methionine amino acids and CPM counts.
Page 12 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 13 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
F- 8. QC plates evaluated during HTP polypeptide library production and purification. Each gel represents 24
samples selected for QC. The IVT reactions were run in 25uL in which 0.25ug of each FTU LEE template was used.
After one hour of incubation, beads were washed and resuspended in SDS loading buffer. Samples were heated to
90°C for 5 minutes and loaded into the wells. These samples contain both the de novo synthesized polypeptides and
the anti-thioredoxin antibody.. After drying, gel was transferred to storage phosphor screen for imaging.
Data locations: Autoradiograph images are stored at:R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU
proteomic library\ 35S QC plates presentation
Sub-milestone 28.3: Array protein-fragments into pools for use as antigen for T cell
stimulation.
After the IVT reactions were completed, supernatants were removed, and products bound to
beads were washed and stored in a final volume of 100ul PBS/ reaction. This bead volume then was split
into two 50ul aliquots. One was used for pooling, and the remaining half-reaction aliquot was stored at 80 oC to be used in a future experiment. To prepare the first half of each reaction for cellular assays at
UNM, polypeptides were pooled by columns 1 through 12 of each 96 well plate. Since the last row (H)
of each plate was left empty, each library plate contained only 7 rows (A to G) of FTU polypeptides. By
Page 13 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 14 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
pooling columns, each pool contains 7 polypeptides and each 96-well plate generated 12 pools. Initially
the total volume of each pool was 350ul (50ul x 7 IVT products). ASU added 50ul of PBS into each pool
to make a total pool volume of 400ul, representing 360ul plus a 10% volume margin in order to
accommodate pipetting loss during distribution. From each 360ul of pooled product, ASU split off 180ul
for UNM’s first polypeptide test with 2 plates (90ul of pooled product/plate) of splenocytes and the other
180ul for testing with 2 plates (90ul of pooled product/plate) of lymph node cells from LVS vaccinated
NHPs. NOTE: All samples were normalized to the amount of beads. The ranges and average amounts of
the full library should be that of the pools.
F-9. Distribution of each polypeptide pool into four aliquots for use in immune cell
assays.
These F.tularensis antigen pools circled in Figure 9 were sent to UNM on January 5, 2009.
The remaining half set of IVT reactions was stored at ASU for the second NHP experiment at UNM.
4.2
Interpretation
The planned objective for this milestone was to implement the high throughput IVT
protocol developed in milestone 26 for the 2,229 polypeptides representing the full FTU
proteome. While ASU carried out numerous tests during protocol development to
provide the highest probability of success, scaling up is rarely straightforward. ASU
conducted multiple pilot and quality control tests of all reagents and procedures used to
ensure successful synthesis of the 2,229 polypeptides to complete Milestone 28. These
Page 14 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 15 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
quality control measures were worthwhile, since ASU was able to efficiently deliver a
high-quality FTU proteomic library to UNM.
4.3
5
Quality Control
All pilot studies described in section 4 represent quality control performed at each step of
the IVT high through put synthesis.
Deliverables Completed
From ASU subcontract deliverables list: On Milestone 28, ASU implemented a high
throughput production system for generating peptides for use in UNM MS 29. ASU
produced and purified 2,229 IVT products representing the full library of FTU polypeptides.
ASU completed the production and delivered the pooled polypeptides to Terry Wu/Rick
Lyons at UNM on 1/6/09.
ASU provided the locations of each peptide in the pooled matrix in the PCR plates in the
xls files entitled:
Short ORF 1 PCR plates (1 to 4)
Long ORF 1 PCR plates (1 to 4)
FTU_R1_Short_ORF2_PCR plates (1 to 3)
Long ORF 2 PCR plates (1 to 4)
Long ORF 3 PCR plates (1 to 4)
Long ORF 4 PCR plates (1 to 4)
Long ORF 5 Plate 1 Gems info
Long ORF 5 Plate 2 Gems info
Bacterial
Strain
Tissue
identifier
# of
vials
Vial
Conce
ntration
# of
block
s
Tissue
Type
Deliverable Reagents
Storage
Date
Storage location*
media
Stored or
(Institution, room, shelf,
Date
etc)
Transferred
*
~ Mass
per block
Date
Storage location*
Stored or
(Institution, room, shelf,
Date
etc)
Transferred
*
Page 15 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 16 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
Deliverable Reagents
RNA/DNA
# of
vials
Vial
Conce
ntration
Storage
media
Date
Storage location*
Stored or
(Institution, room, shelf,
Date
etc)
Transferred
*
Polypeptid
e
#
Conce
ntration
Storage
media
Entire FTU
polypeptide
library
2,229
Date
Stored or
Date
Transferred
*
01-06-09
0.232
mg/ml
PBS
Storage location*
(Institution, room, shelf,
etc)
UNM, BSL3 lab in BRF
G72, -80 freezer,
bottom shelf
ASU, Biodesign Institute
BDB 229, freezer N
REES Probe#75
*The storage location should allow a future researcher to specifically find the stored
reagent. When the “storage location” is equal to the creator’s location, enter the “Date
Stored” in the “Date Stored or Date Transferred” column. When the “storage location”
indicates that the reagent has been transferred to another institution, enter the “Date
Transferred” in the “Date Stored or Date Transferred” column.
Page 16 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 17 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
6
6.1
Accepted Date:9/11/09
Appendices
Appendix 1: Original Data Tables and Figures (those included in the above
sections 3 and 4)
Table/
Figure1
Title
Notebook Location2
(Notebook # and
page numbers)
F-1
E-gel representation of LEE
constructs comprising FTU ORF
expression library
N/A
F-2
NEB IVT Pure system reagent
expresses row A of FTU LEE
amplification PCR plate 2
N/A
F-3
Testing stability of bead-bound
IVT polypeptide samples with
storage
N/A
Electronic
Location2 (Full
Path & File
Name)
R:\GeneVac\FTU
\Contract\Proteo
me\FTU IVT
Data\FTU
proteomic
library\E-gel\Long
ORF 2
R:\GeneVac\FTU
\Contract\Proteo
me\FTU IVT
Data\FTU
gels\FTU HTP
IVT 35S gels\F
tularensis
proteomic
library\NEB IVT
kit testing 10-802 crop 2
R:\GeneVac\FTU
\Contract\Proteo
me\FTU IVT
Data\FTU
gels\FTU HTP
IVT 35S
gels\Testing
bead storage
temperature 2
Page 17 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 18 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
Notebook Location2
(Notebook # and
page numbers)
Table/
Figure1
Title
Electronic
Location2 (Full
Path & File
Name)
Data location:
R:\GeneVac\FTU
\Contract\Proteo
me\FTU IVT
Data\FTU
gels\FTU HTP
IVT 35S gels\F
tularensis
proteomic library\
Testing large
bach of beads
and Ab 10-23-08
crop
F-4
Evaluation of bulk ordered anti-trx
antibody in purification protocol
N/A
F-5
Autoradiograph of polypeptides
generated from gene-built ORF
templates
N/A
R:\GeneVac\FTU
\Contract\Proteo
me\FTU IVT
Data\FTU
gels\FTU HTP
IVT 35S gels\F
tularensis
proteomic
library\BAG\QC
PCR plate 1 and
2
F-6
Coomassie blue stain of unlabeled
FTU polypeptides from gene-built
templates.
N/A
R:\GeneVac\FTU
\Contract\Proteo
me\FTU IVT
Data\FTU
gels\FTU HTP
IVT Coomassie
gels\F tularensis
Library\BAG QC
plate 1 and 2 1125-08 crop
Page 18 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 19 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
Notebook Location2
(Notebook # and
page numbers)
Table/
Figure1
Title
F-7
Coomassie stained gel of IVT
Calmodulin before (1) and after (2)
purification compared to the
protein standards with shown
amounts.
N/A
Data location:
R:\GeneVac\FTU
\Contract\Proteo
me\FTU IVT
Data\FTU
gels\FTU HTP
IVT Coomassie
gels\ gfp conc
curve gray scale
F-8
QC plates evaluated during HTP
polypeptide library production and
purification
N/A
R:\GeneVac\FTU
\Contract\Proteo
me\FTU IVT
Data\FTU
proteomic library\
35S QC plates
presentation
F-9
Distribution of each polypeptide
pool into four aliquots for use in
immune cell assays.
T-1
FTU IVT protein yield calculation
is based on number of methionine
amino acids and CPM counts
N/A
R:\GeneVac\FTU
\Contract\Proteo
me\FTU IVT
Data\FTU
proteomic
library\TCA CPM
to ug\ BAG2WT
QC addition of
thioredoxin tag
11-26-08
1
2
Electronic
Location2 (Full
Path & File
Name)
Use abbreviation “T” for table and “F” for Figure (e.g. T-1 for table 1 or F-3 for figure 3)
If the data location has changed relative to the location reported in the original monthly
Page 19 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 20 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
technical report, then provide both the previously reported data location and the final data location
Page 20 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 21 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
6.2
Accepted Date:9/11/09
Appendix 2: Quality Assessment of Milestone Completion and Report
Assessment Criteria for Milestone Completion
Evaluation of Milestone Completion Report
Has the Milestone Completion Report format
been used and all sections completed,
including Milestone Summary, Milestone
Objectives, Methods Reagents & SOPs,
Salient Original Data Results Interpretation &
Quality Control, Deliverables Completed, and
Appendices?
Does the Milestone Summary include the
milestone’s goals, milestone results, an overall
interpretation of the milestone’s data and
conclusion?
Do Methods, Critical Reagents and SOPs
include summarized methods and details
necessary to re-perform critical experiments?
A list of critical reagents? The completed table
of SOPs?
Are salient negative and positive original data
included in the Milestone Completion Report?
Has the Deliverables Table been completed?
Have the Appendices been completed?
Are the specific original data associations with
experiments clearly annotated in the “Salient
Technical Data” section of the Milestone
Completion report?
Yes No N/A Comment
X
Received location
files on 8/10/09
X
X
ASU proteome
SOP#3 v 2.0
accepted by NIAID on
12/30/09
X
X
X
Appendix 6.1 is
completed. Appendix
6.3.1 is used for the
calculated values for
the control peptides
based on radioactivity
incorporation.
Appendix 6.3.2 lists
the PCR plate files for
the peptide locations
in the plates.
X
Page 21 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 22 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
Assessment Criteria for Milestone Completion
Evaluation of Data included
Yes No N/A Comment
Are the salient original data and results
X
All data included is
included in an organized, easily interpretable
organized well
format?
Is the rationale included?
X
Do tables and figures have legends and
X
original data location annotations?
Is the data interpretation clear?
X
Is the data storage location listed in Appendix
X
1 sufficient for data retrieval in the future? (E.g.
notebook numbers and pages, electronic file
locations including directory paths and file
names). Are prior data locations crossreferenced to final data locations?
Is the data backed up electronically or in
hardcopy notebooks?
Is the data storage location secured either in a
locked fireproof cabinet for hardcopy or on a
server protected by firewall?
Has the data quality been assessed? How
many replicates and how reproducible is the
data? Has statistical analysis been performed
on the data? What quality control has been
utilized by the subcontractor during the data
generation and assessment?
If a protein or peptide has been synthesized,
how has the protein or peptide sequence been
verified? What percentage of the sequences
has been randomly verified?
X
Electronically only
X
X
X
The quality control is
described throughout
the section 4, rather
than just in section
4.3. Stepwise
assessment of the
Quality of product
was essential to the
high throughput
generation of the IVT
products.
There was not
enough material for
protein sequencing.
However, anti-TRX
binding is done by
beads and by
western. Since the
only trx in E. coli
Page 22 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 23 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
Assessment Criteria for Milestone Completion
lysate would have to
come from the IVT
template, this is
verification that we
are looking at the
FTU fusion product
If a genetic mutant has been made, how has
X
the mutation been verified e.g. DNA
sequencing, PCR sequence verification? How
stable is the mutation? How has the impact of
the genetic mutation on the bacterial growth
been assessed? What is the sensitivity of the
assay?
If an aerosol delivery system has been tested,
how reproducible is the delivery to the animal?
Have sufficient animal numbers been tested to
determine reproducibility?
X
If UVA/psoralen treatment kills the bacteria but
leaves them metabolically active, how is killing
assessed? How sensitive is the assessment
of killing? How is expression of bacterial
epitopes determined?
X
Do UNM and the subcontractor agree that the
data supports the scientific interpretation of the
milestone?
Evaluation of Deliverables, as outlined in
the Statement of Work
Have Standard Operating Protocols have been
written by subcontractor, reviewed by UNM,
revised by subcontractor as requested, and
accepted by UNM? The milestone completion
report will not be accepted by UNM until all the
SOPs are accepted by UNM.
Has the Milestone Completion Report been
written by subcontractor, reviewed by UNM,
X
Yes No N/A Comment
X
ASU Proteome SOP
3 v 2.0 was accepted
by NIAID on 12/30/09
X
Page 23 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 24 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
Assessment Criteria for Milestone Completion
revised by subcontractor as requested, and
accepted by UNM?
Has data from the milestone been submitted
X
by the subcontractor, reviewed by UNM, data
presentation revised by the subcontractor as
requested for clarity, and accepted by UNM?
For deliverable reagents, have the minimum
X
UNM and ASU
number of vials, the minimum concentration,
agreed on the
the minimum block size and the minimum
minimum amount of
weight of tissue been mutually agreed by UNM
protein as 1 to 5 ug,
and the subcontractor?
which was sufficient
for the OVA
polypeptide to work in
the ELIspot assays at
UNM.
Have bacterial strains and tissues been
X
banked at the subcontractor’s institution and
backup stocks and aliquots been received by
UNM for long term storage? A minimum
number of vials of -20C /-80C bacterial stocks
at specified concentration in glycerol are
stored at both institutions. A minimum size
paraffin block or minimum weight of
cryopreserved frozen tissues are stored at
both institutions.
Evaluation of SOPs
Do SOPs contain standard sections e.g.
purpose, list of supplies and equipment
required including vendors and model
numbers, reagent preparation, method, results
expected, description of data interpretation,
criteria for accepting or rejecting results,
description of data storage location, date SOP
is in service, names of people who prepared
and reviewed the SOP?
Can an independent scientist read and
understand the standard operating procedure?
Yes No N/A Comment
X
X
Page 24 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 25 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
6.3
6.3.1
Accepted Date:9/11/09
Appendix 3: Additional Data/Figures NOT included in the Text of the Milestone
Completion Report (Section 3 or 4)
Insert : Example protein yield calculation
FTU0029C: 39048.4 Daltons molecular weight, 6 radiolabeled methionine residues in
the peptide
Total methionine= 7500pmol (unlabeled) + 8.68 pmol (radiolabeled)=7508.68pmol
CPM’s Measured: 10420 cpm total in a 2.5ul aliquot, which is 10% of the total reaction
volume of 25 ul
106 cpm background
3638cpm TCA ppt (incorporated into protein)
Total count in the reaction= (10420cpm/2.5uL) x 25uL= 104,200 cpm
Specific activity of methionine in the reaction= (104200 total cpm/7508.68 pmol)=13.88
cpm/pmol
Methionine incorporation= [(3638cpm-106cpm) x10]/13.88 cpm/pmol= 2546.6 pmol
Met
pmoles FTU0029C= 2546.6 pmol Met/ 6 Met per protein= 424.4pmol
Yield (ug)= (424.4pmol x 39048.4 daltons)/106= 16.57ug protein made/25ul rxn
Note: 1 Kda protein will have a concentration of 1Kg/mol. Then you convert mole to
picomole and Kg to microgram.
6.3.2
ASU provided the locations of each peptide in the pooled matrix in the xls files entitled:
Short ORF 1 PCR plates (1 to 4).xls
ASU MS28 Long ORF 1 PCR plates (1 to 4).xls
ASU MS28 FTU_R1_Short_ORF2_PCR plates (1 to 3).xls
Page 25 of 26
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS # 28
Institution: Arizona State University
Author: Kathryn Sykes
MS Start
Date:3/1/2007
MS End
Date:12/31/2008
Report Date: 3/4/2009
Version: 2.0
Page 26 of 26
Reviewed by : Barbara Griffith 4/24/09, 7/22/09,
8/10/09, 9/11/09, 10/27/09, 12/23/09, 1/4/10, 1/6/10
Accepted Date:9/11/09
ASU MS28 Long ORF 2 PCR plates (1 to 4).xls
ASU MS28 Long ORF 3 PCR plates (1 to 4).xls
ASU MS28 Long ORF 4 PCR plates (1 to 4).xls
ASU MS28 Long ORF 5 Plate 1 Gems info.xls
ASU MS28 Long ORF 5 Plate 2 Gems info.xls
Page 26 of 26