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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 1 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Signature Page
Author’s Signature:
______Ping Chu / Jirong Liu_________
Typed Name of Author
__Not required_______________________ __________
Signature of Author
Date Signed
Acceptance by Subcontracting Institution:
_Karl Klose_______________________
Typed Name of Subcontracting PI
_ Not Required____________________
Signature of Subcontracting PI
__________
Date Signed
Acceptance by the University of New Mexico:
________________________________
C. Rick Lyons, MD. PhD
Not Required_______________________ __________
Signature
Date Signed
Acceptance by NIAID:
__Freyja Lynn____11/05/2008 accepted
Typed Name of NIAID Interim Project Officer
Not required________________________ _HHolley CO accepted 1.26.09
Signature of NIAID Project Officer
Date Signed
1 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 2 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Table of Contents
1
2
3
4
Milestone Summary ...................................................................................................................................................2
Milestone Objectives..................................................................................................................................................3
Methods, Critical Reagents and SOPs........................................................................................................................3
Salient Original Data, Results, Interpretation, Quality Control ............................................................................... 25
4.1
Original Data and Results (Rationale, Tables/Figures with legends and location annotations) ............... 25
4.2
Interpretation ............................................................................................................................................ 25
4.3
Quality Control ......................................................................................................................................... 25
5 Deliverables Completed ........................................................................................................................................... 26
6 Appendices ............................................................................................................................................................... 29
6.1
Appendix 1: Original Data Tables and Figures ....................................................................................... 29
6.2
Appendix 2: Quality Assessment of Milestone Completion and Report ................................................. 33
6.3
Appendix 3: Additional Data/Figures not included in the Text of the Milestone Completion Report
(Section 4) .............................................................................................................................................. 37
1
Milestone Summary
1.1 One of the goals of the milestone was to create uvrA and uvrB mutants of F. tularensis subsp.
Holarctic LVS. UTSA successfully created the uvrA and uvrB mutants of F. tularensis subsp.holarctic
LVS. The LVS uvrA and uvrB mutants will be used by Cerus in hopes of making a killed but
metabolically active (KMBA) vaccine. Mutation of the LVS uvrA and uvrB genes was expected to
impair DNA repair, potentially inhibit bacterial replication, and yet maintain metabolic activity in the
Cerus KBMA approach. The overall goal of the TVD contract is to identify and characterize potential
tularemia vaccines, and this milestone contributes to the evaluation of KBMA vaccines as a potential
tularemia vaccine.
1.2 The other goal was to create a T-cell epitope tagged protein which was expressed by F. tularensis
within host cells. The only well-characterized secreted Ft protein was PepO, and the T cell tag was
SIINFEKL. UTSA successfully created the plasmid carrying PepO- SIINFEKL which was expressed by
F.tularensis subsp.novicida U112 and holarctic LVS. U112 and LVS with PepO-SIINFEKL will be
used by Cerus. Cerus requested the construction of a F.tularensis secreted protein fused to the T cell
epitope SIINFEKL, in order to study MHC-I processing in F.tularensis-infected cells. SIINFEKL is
presented to T cells via MHC-I, and thus using F.t.-infected cells expressing PepO-SIINFEKL, Cerus
can study T cell responses to these cells.
2 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Version: 1.0
Page 3 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
2
Accepted
Date:10/22/08
Milestone Objectives
2.1 UTSA will create uvrB or uvrA mutant in F.tularensis subsp.holarctic LVS.
2.2 UTSA will construct the plasmid with PepO-SIINFEKL and transform the plasmid into F.tularensis
subsp.novicida U112 and holarctic LVS. (This was an additional goal added after the UTSA subcontract
was signed in Spring 2006)
2.3 Cerus will “examine the KBMA F novicida mutants agreed upon”. Cerus’ Milestone #43 was
terminated before it began. See the explanation in the MS#43 MSCR deliverables section, to follow.
3
Methods, Critical Reagents and SOPs
Methods for uvrB mutant in LVS
3.1 Method 1-Primers used to amplify uvrB upstream, downstream and Kanamycin resistant marker are:
For uvrB LVS upstream fragment:
uvrBLVSup: 5’gga gaa ttcc gc agc aga tga tat tgc acg cac a
uvrBDn: 5’ act act ggg ctg ctt cct aat gca ttg tat tgc ttg agg ctg atc gcc
For uvrB LVS downstream fragment:
uvrBup1: 5’ gct gct aac aaa gcc cga aag gaa gct acg aag gtt atc aaa gct ctc g
uvrBLVSdn1: 5’ gga gaa ttc ttg cac caa tcc cgg caa gtaa
For Fp-Kanamycin resistant marker:
KanFNdeI: 5’ gga att cca tat gag ccca tat tca acg ggaa
KanRBamHI: 5’ cgc gga tcc tta gaa aaa ctc atc gag cat caa atg
This method was to amplify the UvrBFpKan fragment using overlapping PCR with UvrB upstream
fragment, FpKan fragment and UvrB downstream fragment for the templates, and UvrBLVSUp and
UvrBLVSDn1 for the primers. Since the PCR product was not as expected, a new ”method 2” was
used. Data are recorded on TVDC UTSA Notebook#2, page50-52.
3.2. Method 2-The individual fragment of uvrBLVS up & downstream was cloned into pUC19 plasmid
and transformed into E.coli.TOP10. The plasmid pKEK1028 (uvrBLVS upstream) and
pKEK1029 (uvrB LVS downstream) were constructed. Upstream, downstream and FpKan (in
pKEK898) fragments were released after being cut with certain restriction enzymes. Then three
fragments were ligated together, and transformed into LVS, but no positive colonies were
observed.
The primers used to amplify uvrB LVS upstream and downstream fragments were:
For upstream seq.:
uvrBLVSUp: 5’ gga gaa ttc gca gca gat gat att gca cgc aca
uvrBLVSdn: 5’ gga gga tcc ttg tat tgc ttg agg ctg atc gcc
For downstream seq.:
3 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 4 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
uvrBLVSup1: 5’ gga gga tcc gct acg aag gtt atc aaa gct ctc g
uvrBLVSdn1: 5’ gga ctg cag tca gca gat gat att gca cgc aca
Data recorded on TVDC UTSA Notebook#2, pages52-54.
3.3 Method 3-Since the initial two methods were used without successfully transforming a uvrB
construct into LVS, a third technique was tried. A new plasmid was created based on the
backbone pDS132 which contains sacB counter selectable marker and Chloramphenical
resistance marker to mate into LVS to generate a uvrB mutant in LVS.
Mutagenesis vector pDS132 was modified to:
3.3.1 Contain more useful restriction sites in MCS (Multiple cloning site)
The primers used to amplify pDS132 backbone are:
pDS132F: 5’ gga tcc ctg cag tgc taa tct ggg ccc gcg gcc gcg acg tcg tcg act gga aga
agc aga ccg cta aca
pDS132R: 5’ gga tcc ctg cag acg cgt tcg agt cta gac ata tgg ata tca gct ctc ccg gga attc
The PCR fragment was cut with PstI , re-ligated and transformed into DAPA- cells. Screened the
CmR (chloramphenical resistant) colonies by restriction digestion with XhoI and NcoI.
3.3.2 Contain Ft groELp (Fransicella groEL gene promoter) to drive sacB and CmR expression.
GroELp promoter was released from pKEK842 with SalI and NotI restriction digestion, and the
released fragment was ligated into the new pDS132 from step3.3.1 cut with the same enzymes,
then electroporated into DAPA- cells. The potential CmR colonies were screened by colony PCR
with pGroELpdown and SacBCDSR primers and a correct colony was identified
The modified pDS132, which contained the CmR and Ft groELp, was renamed pKEK1090.
3.3.3 The plasmid pKEK1006 was cut with NotI to release uvrBFpKan which was then ligated
into pKEK1090 cut with NotI. The ligated fragment was electroporated into DAPA- cells, and
plated onto LB/DAPA/Kan plate. The Kan resistant colonies were grown on LB/DAPA/Cm plate
to make sure they were not from carryover plasmid of pGEM-T FpKanuvrB. This new
construction was named pKEK1114.
3.3.4 Conjugated pKEK1114 into LVS at 1: 10 ratio, isolated co-integrants by selection for CmR,
Cm resistant colonies were selected by Kan (Kanamycin) and SacB (Sucrose sensitivity). PCR
indicated these KanR and SacBR colonies were correct co-integrants.
3.3.5 Counter selection for second recombination was attempted on TSA+++ 5% sucrose (+ Kan)
and this did not result in loss of co-integrated plasmid. Also several different media formulations
were tested with 5% sucrose, but none allowed growth of LVS. So a higher concentration of
sucrose was used next to try to remove the plasmid backbone but maintain the interruption of the
uvrB gene in LVS.
4 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 5 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
3.3.6 LVS with co-integrated uvrB::Kan plasmid was grown on TSA+++/10% sucrose/ Kan plate
to try to remove the plasmid. Patched the colonies from TSA+++/Kan/10%Sucrose plate on both
TSA+++/Kan plates and TSA+++/CM plates. Kan resistant, but Cm sensitive colonies were
considered the correct ones which were the expected phenotype of strains that had lost plasmid
(CmS), but had retained uvrB::Kan mutation (KanR).
3.3.7 The primers used to screen LVS uvrB::Kan colonies were:
For uvrBFpKan seq.:
uvrBLVS PstI up: 5’ gga ctg cag gca gca gat gat att gca cgc aca
uvrBLVS PstI Dn1: 5’ gga ctg caag ttg cac caa tcc cgg caa gta a
For SacB seq:
SacBCDSDnBgl2: 5’ gga aga tct tta ttt gtt aac tgt taa ttg tcc
SacBPUpBamHI: 5’ cgc gga tcc cca tct tca aac agg agg gct gga
3.3.8 Set up following PCR reaction to amplify uvrBFpKan seq.:
dd water
34.0ul
10x XL buffer
5.0ul
KOD dNTPs
5.0ul
DNA Template
1.0ul
UvrBLvsPstlUp primer
2.0ul
UvrBLvsPstlDn1 primer
2.0ul
XL DNA polymerase
1.0ul
At 94C 1min, then 94C 30 sec/60C 10sec/ 72C 6 min// 30 cycles, then 72C 10 min
Figure1: The gel picture of UvrBFpKan PCR .
1
2
3
4
5
6
7
8
9
1.Marker (1kb ladder)
2.UvrBFpKan U112
3.Wild type LVS
4.sample 6
5.sample 7
6.sample 8
7.sample 9
8.sample 6
9.Marker
Figure 1 title, legend and data location: Amplification of UvrB gene from the potential UvrB mutant LVS
chromosomal DNA. This PCR was performed to screen the mutant uvrB LVS using UvrBLvsPstlUp and
5 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 6 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
UvrBLvsPstlDn1 primers. Lane4-7 (colony6-9) were four potential uvrB mutant LVS which presented PCR
products (about 4.0kb) a little bit larger than wt LVS (lane3, about 5.0kb). This indicated that these four Kanamycin
resistant colonies might be the expected mutants. Data are located in Notebook #2, page 67.
3.3.9 Set up following PCR reaction to amplify SacB seq:
dd water
32.6ul
10xbuffer #1 for KOD
5.0ul
KOD dNTPs
5.0ul
MgCl2
2.0ul
DNA Template
1.0ul
SacBCDSDnBgl2 primer
2.0ul
SacBPUpBamHI primer
2.0ul
KOD HiFi DNA polymerase
0.4ul
At 98C 1min, then 98C 15 sec/48C 15sec/ 72C 1min// 30 cycles
Figure2: The gel picture of SacB PCR.
Figure 2 title, legend and data location: Amplification of SacB gene from the potential UvrB mutant LVS. This
PCR was amplified to verify the existence of the plasmid carrying SacB in potential UvrB mutant LVS. Lane2 was
the positive control of SacB (about 1.4kb), whereas there was not any band in lane3-lane6 (colony6-9), which gave
the evidence that the plasmid backbone was removed. Data are located in TVDC UTSA Notebook#2, pages 68.
6 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 7 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
3.3.10 Digestion with Bgl2 restriction enzyme for gel purified DNA from Figure1.
Figure3: The gel picture of digestion of UvrBFpKan PCR gel purified DNA with Bgl2.
1
2
3
4
5
6
7
1.Marker
2.UvrBKanU112 cut
3.UvrBKanU112 uncut
4.LVS cut
5.Sample 6 cut
6.Sample 6 uncut
7.Marker
Figure 3 title, legend and data location: Digestion of mutant UvrB gene from UvrB mutant LVS with Bgl2. Lane2
was the positive control digested with Bgl2, and showed two bands afterwards. Lane3 was the same sample as the
positive control but without being digested. Lane 4 was the negative control (wild type LVS) treated with Bgl2, but
nothing happened since there was no Bgl2 restriction site in wt LVS. Lane5 was the potential UvrB mutant LVS cut
with Bgl2, and two fragments were presented after being digested compared to lane6, which was the same sample
without being treated with Bgl2. This result gave the concrete evidence that uvrB in LVS had been mutated for the
colony we analyzed. Data are recorded on UTSA TVDC notebook #2, page 67 for Figure3.
3.3.11 Gel purified DNA from Figure1 was sent for sequencing with KanFNdeI and KanRBamHI
primers, and the result confirmed LVS uvrB::Kan was correct. LVS uvrB::Kan was named as
KKF303. KKF303 is a deliverable uvrB mutant in LVS.
Sequencing Data recorded on UTSA TVDC notebook#2, page69.
3.3.12 Method 3 successfully created the KKF303, which is a deliverable uvrB mutant in LVS.
Methods for uvrA mutant in LVS
3.4 Strategy 1: The strategy for this method was to conjugate the plasmid pKEK1120, which
contained uvrAFpKan and the GroEL promoter properly inserted to facilitate sacB/Cm
expression, into LVS. SacB was expected to help eliminate the plasmid backbone by the
7 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 8 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
counterselection on sucrose. LVS and E.Coli./DAPA-/pKEK1120 were grown on agar plates for
about 4-6 hours for LVS and 2-3 hours for E.coli, then mixed the bacterial colonies at an
approximate ratio of LVS:E.Coli.=10:1. The mixture was grown on TSA++/DAPA plate for
overnight, and transferred onto TSA++/Cm plate to grow for 4-10days. Then CmR colonies were
grown on TSA++/Kan plate. This procedure was repeated for several times. Either no colonies
were observed on the plate, or the colonies were the correct co-integrants as determined by
colony PCR, but the colonies were hardly able to survive or grow on agar plates or in liquid
medium. Since it didn’t work out for us to use this strategy after we tried several times, there was
a new strategy to disturb and inactivate the gene by insertion mutagenesis in the target gene. This
new strategy worked successfully in our lab and we decided to switch the strategy from the
conjugation to the targetron gene knockout system to create the UvrA mutant in LVS.
3.4.1 The primers used to amplify Kan seq. in the co-integrants by colony PCR are:
KanFNdel : 5’ gga att cca tat gag cca tat tca acg gga a
KanRBamH1: 5’ cgc gga tcc tta gaa aaa ctc atc gag cat caa atg
3.4.2 Set up following colony PCR :
dd water
32.6ul
10xBuffer#1 for KOD 5.0ul
MgCl2
2.0ul
dNTPs
5.0ul
KanFNdel
2.0ul
KanRBamH1
2.0ul
KOD Hifi polymerase 0.4ul
DNA
1.0ul
At 98C 1min, then 98C 15 sec/55C 15sec/721min//30cycles
Figure4: The gel picture of Kanamycin PCR.
1
2
3
4
1 1kb Marker
2 UvrBKan U112
3 Colony1
4 Colony2
8 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 9 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Figure 4 title, legend and data location: Amplification of Kanamycin selection marker gene from the potential
UvrA mutant LVS colony. This PCR was to identify the existence of Kanamycin gene in UvrA mutant LVS using
KanFNdel and KanRBamHI primers. Lane2 was the positive control with the band about 700bp. Lane3 (colony1)
and lane4 (colony2) had the same band as the positive control, which indicated that UvrA in LVS might be mutated
in presence of Kanamycin. Since either of potential UvrA mutant LVS was able to survive, we could not to screen
them for further confirmation. Data are recorded on UTSA TVDC notebook #2, page74 for figure4.
3.5 Strategy 2: Since we could not conjugate pKEK1120, , which contained uvrAFpKan and the GroEL
promoter properly inserted to facilitate sacB/Cm expression, into LVS or keep the strain alive
afterwards successfully, we applied a new strategy using the Targetron Gene Knockout System
to create uvrA mutant in LVS. The Targetron vector, designed to be specific for the uvrA gene,
was introduced into LVS to re-target and inactivate the uvrA gene.
3.5.1 The primers used to amplify 350bp mutated intron RNA PCR fragment are:
FTT1312c-72/73s-IBS: 5’ aaa act cga gat aat tat cct taa tac ccc ggg atg tgc gcc cag tat ggg tg
FTT1312c-72/73s-EBS1d: 5’ cag att gat caa atg tgg tga taa cag ata agt ccg gga taa taa ctt acc
ttt ctt tgt
FTT1312c-72/73s-EBS2: 5’ tga acg caa gtt tct aat ttc gat tgg tat tcg ata gag gaa agt gct t
FTT1312c-259/260a-IBS: 5’ aaa act cga gat aat tat cct taa tgt tct ttt ttg tgc gcc cag ata ggg tg
FTT1312c-259/260a-EBS1d: 5’ cag att gta caa atg tgg tga taa cag ata agt ctt ttt tga taa ctt acc
ttt ctt tgt
FTT1312c-259/260a-EBS2: 5’ tga acg caa gtt tct aat ttc gat taa cat tcg ata gag gaa agt gtc t
EBS Universal: 5’ cga aat tag aaa ctt gcg ttc agt aaa c
3.5.2 Set up following PCR reaction to amplify the 350bp PCR seq.
23ul ddH2O
1.0ul 4-primer mix ( IBS, EBS1d, EBS2 and EBS Universal )
1.0ul Intron PCR template
25.0ul JumpStart RED taq Ready mix
AT 94C 30sec, 94C 15sec/55C 30sec/72C 30sec//30 cycles, then 72C 2min
9 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 10 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Figure 5: The gel picture of 350bp PCR product.
1
2
3
4
5
1.0kb
1.PCR1
2.PCR2
3.PCR3
4.PCR4
5.1kb ladder
0.5kb
Figure 5 title, legend and data location: Amplification of 350bp PCR product for re-targeting the intron RNA.
This process was to amplify PCR to mutate (re-target) intron RNA (350bp PCR product). There were three bands
shown on the gel. The upmost band was the 350bp PCR product. Data are located in TVDC UTSA notebook#2,
page 76.
3.5.3 The 350bp mutated intron RNA PCR (at 72/73s retarget site) fragment was gel purified.
The purified PCR DNA and the plasmid pKEK1140, which was modified as an intron expression
vector and the backbone of the Targetron vector, were double digested with XhoI and BsrGI, then
two digested fragments were ligated and transformed into E.Coli. DH5 by electroporation.
3.5.4 The potential transformants (white colonies) from LB /X-gal/ Kan plate were screened by
digestion with BglII at 37C for 3 hours. The figure for the digestion as follows:
10 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 11 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Figure 6: The gel picture of digestion of pKEK1140/350bp PCR fragment with Bgl2.
01 02
03 04
05
06
07
08
09
10
11
12
13 14
4.0kb
3.0kb
0
01) 1kb marker
02) colony1(uncut)
03) colony1(cut)
04) colony2(cut)
05) colony3(cut)
06) colony4(cut)
07) pKEK1140(uncut)
08) pKEK1140(cut)
09) colony5(uncut)
10) colony5(cut)
11) colony6(cut)
12) colony7(cut)
13) colony8(cut)
14) 1kb marker
Figure 6 title, legend and data location: Digestion of the potential Tulatron vector DNA with Bgl2.
Lane7 was the parent plasmid pKEK1140without being treated with Bgl2, and lane8 (pKEK1140) was the positive
control digested with Bgl2 (3 fragments after being cut). Lane3-6 were colony1-4 cut with Bgl2, and lane10-13 were
colony5-8 cut with Bgl2, All potential transformants (except for colony 6) gave the correct digestion pattern and
were correct compared to the parent plasmid pKEK1140. The difference between the parent pKEK1140 and the
mutants was that the uppermost band was a little bit more than 4.0kb in parent plasmid and a little less than 4.0kp in
mutant plasmid after being digested with BglII. Data are recorded on UTSA TVDC notebook #2, page 78.
This new plasmid was designated as pKEK1167 (at 72/73 target site).
3.5.5 The plasmid pKEK1167 was transformed into wild type LVS by electroporation. The
transformed cells were plated onto TSA++/ Kan plate, and incubated at 30C for 3-4 days to get
the single colonies. The potential transformants were screened by colony PCR.
3.5.6 The primers used for colony PCR were:
UvrASchu4Up (flanking uvrA in LVS): 5’ gga gaa ttc tga agc tat agc aga ggc tcg tga
UvrASchu4LVSDn(flanking uvrA in LVS):5’ act act ggg ctg ctt cct aat gca aca aca tac ctt ctt tgc
cct tca gc
EBS Universal (in intron RNA): 5’ cga aat tag aaa ctt gcg ttc agt aaa c
11 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 12 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
3.5.7 Set up following colony PCR reaction to confirm the intron insertion in UvrA gene and the
orientation of insertion:
32.6ul ddH2O
5.0ul 10XBuffer #1 for KOD
5.0ul dNTPs
2.0ul MgCl2
1.0ul DNA
2.0ul UvrAShcu4Up
2.0ul EBS Universal
0.4ul KOD HiFi polymerase
At 98C 1min, 98C 15sec/ 55C 15sec/ 72C 1min// 30 cycles
34.0ul ddH2O
5.0ul 10XKOD XL Buffer
5.0ul dNTPs
1.0ul DNA
2.0ul UvrASchu4LVSDn
2.0ul EBS Universal
1.0ul KOD XL DNA polymerase
At 94C 1min, 94C 30sec/ 55C 10sec/ 72C 2min// 30 cycles, 72C 10min
Figure 7: The gel picture of colony PCR to confirm the intron insertion in UvrA gene of LVS.
1
2
3
4
5
6
7
8
9 10 11 12
1.1kb ladder
2.Colony 1
3.Colony 2
4.Colony 3
5.Colony 4
6.Wild type LVS
1.0kb
7.Colony 1
8.Colony 2
0.5kb
9.Colony 3
p
10.Colony 4
11.wt LVS
12.1kb ladder
Lane 2-6 PCR with UvrASchu4Up and EBS Universal primers
Lane 7-11 PCR with UvrASchu4LVSDn and EBS Universal primers
Figure 7 title, legend and data location: Colony PCR for confirmation of the intron insertion in UvrA gene of
LVS. UvrASchu4Up and UvrASchu4LVSDn were the UvrA gene specific primers flanking the intron insertion, and
EBS Universal was the intron insertion specific primer. Lane6 and 11 (wild type LVS) were the negative controls.
Lane2-5 (colony1-4) and lane6 were PCRs amplified with UvrASchu4Up and EBS Universal primers. The bands
12 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 13 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
shown on lane2-5 were about 600bp, which was expected. Lane7-10 (colony1-4) and lane11 were amplified with
UvrASchu4LVSDn and EBS Universal primers, which didn’t produce any specific band. Data are located in TVDC
UTSA notebook#2, page 80.
The mutant intron RNA was in uvrA of LVS, which would result in the mutation of the uvrA
gene in LVS.. UvrASchu4Up was in the upstream of UvrA, whereas UvrASchu4LVSDn was
located in the downstream of UvrA. EBS Universal primer was in the reverse direction of
uvrASchu4up. There was no specific PCR product for primers UvrASchu4LVSDn and EBS
Universal, which meant the two primers were in the same orientation.
3.5.8 Set up following colony PCR with UvrASchu4Up and UvrASchu4LVSDn primers to further
confirm the insertion.
32.6ul ddH2O
5.0ul 10XBuffer #1 for KOD
5.0ul dNTPs
2.0ul MgCl2
1.0ul DNA
2.0ul UvrAShcu4Up
2.0ul UvrASchu4LVSDn
0.4ul KOD HiFi polymerase
At 98C 1min, 98C 15sec/ 55C 15sec/ 72C 1min30sec// 30 cycles
Figure 8: The gel picture of colony PCR using the primers flanking the intron insertion.
1
2.0kb
1.5kb
1.0kb
0.5kb
2
3
4
5
6
1.1kb ladder
2.Colony1
3.Colony2
4.Colony3
5.Colony4
6.Wild type
LVS
Figure 8 title, legend and data location: Colony PCR for confirmation of the intron insertion in UvrA gene of
LVS. Lane 6 was the negative control (wt LVS), which should have one specific band at about 600bp. Lane2-lane5
were colony1-colony4, which had two specific bands on colony1-3 and one band on colony4. Data are located in
TVDC UTSA Notebook#2, page 81
This PCR confirmed the insertion into the UvrA gene of LVS. For the insertion in UvrA of LVS,
PCR product with two primers flanking the intron insertion should be larger (about 1300bp) than
wild type LVS (about 600bp PCR product). There were two bands for colony 1,2 and 3. The
smaller band was about the same size (600bp) as wild type LVS, and the larger band was about
1300bp. It was possible that LVS had the plasmid inside but no insertion happened during the
13 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 14 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
transformation and incubation afterwards. Colony4 (lane5) was probably the pure UvrA mutant
LVS with insertion in uvrA gene because only the larger band was present.
3.5.9 The gel purified DNA from PCR product with UvrASchu4Up and EBS Universal primers
was sent for sequencing with the same primers, and the sequencing result confirmed that the
insertion was in UvrA(1312C) at 72/73bp in LVS.
Sequencing data recorded on UTSA TVDC notebook #2, page 84.
3.5.10 To separate LVS with the insertion in the uvrA gene from LVS without insertion in the
uvrA gene, colony4 was streaked onto TSA+++/ Kanamycin(50ug/ul) plate and incubated at
30C to get single colonies. Then colony PCR was performed with the same primers as 3.5.7 and
3.5.8 to screen the colonies until the colony with the insertion was separated completely from the
colony without insertion. All incubation was done at 30C.
3.5.11 Since the plasmid was temperature sensitive and resistant to Kanamycin, to remove the
plasmid from the UvrA mutant LVS, the UvrA mutant LVS with plasmid was streaked onto
TSA+++/ Ampicillin (100ug/ul) plate and incubated at 37C to get single colonies. Then the
single colonies were grown onto TSA+++/ Ampicillin( (100ug/ul) and TSA+++/
Kanamycin(50ug/ul) plates, and incubated at 37C. The same procedure was repeated until we
got Kan sensitive and Amp resistance colonies. All incubations were done at 37C.
3.5.12 Performed the colony PCR with UvrASchu4Up and EBS Universal, UvrASchu4Up and
UvrASchu4LVSDn primers for the Kanamycin sensitive colony with the same settings as 3.5.7 and
3.5.8
14 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 15 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Figure 9: The gel picture of colony PCR for UvrA mutant LVS with the plasmid being
removed using UvrASchu4Up and EBS Universal, UvrASchu4Up and
UvrASchu4LVSDn primers
1 2 3 4
5 6 7 8 9 10
1.0kb
0.5kb
1.5kb
1.0kb
0.5kb
1.1kb ladder
2.Positive cotrol
3.Wild type LVS
4.Colony2
5.Colony 3
6.Colony 13
7.Colony 21
8.Colony 24
9.Colony 27
10.1kb ladder
11.1kb ladder
12.Positive control
13.Wild type LVS
14.Colony 2
15.Colony 3
16.Colony 13
17.Colony 21
18.Colony 24
19.Colony 27
20.1kb ladder
11 12 13 14 15 16 17 18 19 20
Figure 9 title, legend and data location: Colony PCR for confirmation of the UvrA mutant LVS with the
plasmid being removed. Data located in TVDC UTSA notebook#2, page 83.
Lane 2-9: Colony PCR with UvrASchu4Up and EBS Universal primers (approximately 600bp)
Lane12-19: Colony PCR with UvrASchu4Up and UvrASchu4LVSDn primers (approximately 1300bp)
Lane2 and 12 were the positive controls, and land3 and 13 were the negative controls (wt LVS). All the
colonies (lane4-9 and lane14-19) had the same bands as the positive controls. Data recorded on UTSA TVDC
notebook #2, page 79-83 for Figure7-9.
3.5.13 This colony PCR indicated that the mutated intron RNA was inserted in UvrA(1312C) at
72/73 bp of LVS and the plasmid had been removed. This UvrA mutant LVS was Named KKF317.
KKF317, the uvrA mutant in LVS, is a deliverable on Milestone 43.
3.5.1.4 This UvrA mutant LVS was Named KKF317. KKF317, the uvrA mutant in LVS, is a
deliverable on Milestone 43.
Methods for PepO-SIINFEKL in LVS
3.6 A new plasmid was created to combine PepO-SIINFEKL with the backbone pKEK1145.
The
plasmid pKEK1145 was constructed with the plasmid pBAD24 for the backbone and expressing PepOFlag. A pair of complimentary oligonucleotides encoding SIINFEKL was used to replace the FLAG tag
fragment in pKEK1145. The method for this strategy follows below.
15 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 16 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
3.6.1 The primers used to amplify SIINFEKL fragment were:
TCell tag for: 5’-TCG AGT CAA TAA TAA ATT TCG AAA AGC TTT AGC TGC A-3’
TCell tag rev: 5’-GCT AAA GCT TTT CGA AAT TTA TTA TTG AC-3’
3.8 Set up following PCR reaction for SIINFEKL seq.
1ul TCell tag for (100pmol/ul)
1ul TCell tag rev (100pmol/ul)
18ul DNA Bind Buffer
At 96ºC 1min, then 0.1ºC/s to 4.0ºC
Figure 10: The gel picture of PCR for SIINFEKL fragment.
1
2
3
1.100bp Ladder
2.PCR1
3.PCR2
100bp
Figure 10 title, legend and data location: Amplification of SIINFEKL fragment. The expected PCR product
should be about 37bp. Lane2 and 3 showed the expected bands. Data located in TVDC UTSA Notebook#2, pages
104.
3.6.2 The plasmid pKEK1145 was digested with XhoI and PstI individually and purified with gel
extraction kit. SIINFEKL fragment was ligated into digested pKEK1145, and transformed into
E.Coli. DH5. The transformed cells were plated onto LB/Amp plate and incubated at 37C for
overnight.
16 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 17 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
3.6.3 The plasmid DNA purified from the transformants was shown on figure11. The mutant
pKEK1145 should be about the same size as the parent plasmid, so colony 7 and colony 9 were
not correct.
Figure 11: The gel picture of the potential transformants plasmid DNA.
01 02 03 04 05 06 07 08 09 10 11 12
13
01) 1kb ladder
02) colony 1
03) colony 2
04) colony 3
05) colony 4
06) colony 5
07) pKEK1145
08).colony 6
09) colony 7
10) colony 8
11) colony 9
12) colony 10
13) 1kb ladder
Figure 11 title, legend and data location: The miniprep DNA of the transformant pKEK1145 with SIINFEKL.
Lane2-lane6 were the potential transformants #1-#5, and lane8-lane12 were #6-#10. #7 and #9 were smaller than the
control pKEK1145. Except for #7 and #9, all other tranformants might be what we expected. Data are located in
TVDC UTSA Notebook#2, page 106.
3.6.4 The primers used for colony PCR to confirm the replacement of Flag-tag with SIINFEKL
tag were:
PepO For (in PepO seq.): 5’ gcg gcg ccc gct tat aaa cta tct tta aat gg
SIINFEKL Rev (in SIINFEKL seq.): 5’ cgc gct gca gct aaa gct ttt cga aat
3.6.5 Set up following colony PCR to confirm the insertion:
32.6ul ddH2O
5.0ul 10xBuffer#1 for KOD
5.0ul KOD dNTPs
2.0ul MgCl2
1.0ul mini prep DNA
2.0ul PepO For primer
2.0ul SIINFEKL Rev primer
0.4ul KOD HiFi DNA polymerase
At 98ºC 1min, 98ºC 15sec/57ºC15sec/72ºC 1min//30cycles
17 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 18 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Figure 12: The gel picture of colony PCR for pKEK1145/SIINFEKL to verify the existence of
SIINFEKL
.
01
02
03
04
05
06
07
08
09
10
01).1kb marker
02) Colony 1
03) Colony 2
04) Colony 3
05) Colony 4
06) Colony 5
07) Colony 6
08) Colony 8
09) Colony 10
10) pKEK1145
Figure 12 title, legend and data location: Colony PCR for confirmation of SIINFEKL in pKEK1145. One of the
primers “PepO For” was in the parent plasmid pKEK1145 and the other one “SIINFEKL Rev” was in SIINFEKL.
Lane10 (pKEK1145) was the negative control. Lane2-9 (8 transformants) had the specific band (about 300bp) on
each lane. Data are located in TVDC UTSA Notebook#2, page 107
The PCR product (about 300bp, lane2-lane9) confirmed that the insertion SIINFEKL was in
pKEK1145 So in comparison, the Flag-tag was replaced with the SIINFEKL tag in the parent
plasmid pKEK1145.
3.6.6 Since pKEK1145 didn’t contain Francisella promoter which could facilitate PepO-
SIINFEKL expression in Francisella, the PepO-SIINFEKL construct must be released from
pKEK1145 plasmid backbone and ligated into a different plasmid containing the Francisella
promoter. The plasmid pKEK1149 contained F. promoter and expressed PepO-Flag. It could be
transformed into Francisella to drive PepO-SIINFEKL expression directly after Flag-tag fragment
was replaced with SIINFEKL tag. The exact same procedures as were used for the pKEK1145
were applied to replace Flag-tag fragment with SIINFEKL in pKEK1149, and only one
transformant was observed. The same primers and PCR reaction as step 4.2 were used for this
colony PCR. It proved that it was correct.
Figure 13: The gel picture of PCR for pKEK1149/SIINFEKL using PepO For and SIINFEKL Rev
primers.
18 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Version: 1.0
Page 19 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
1
1kb
2
3
Accepted
Date:10/22/08
4
1 1kb ladder
2 pKEK1149(SIINFEKL) mini prep
3 pKEK1145(SIINFEKL) mini prep
4 pKEK1149
0.5kb
Figure 13 title, legend and data location: PCR for confirmation of SIINFEKL in pKEK1149
Lane4 (the parent plasmid pKEK1149) was the negative control and lane3 (pKEK1145/SIINFEKL) was the positive
control (about300bp). The potential new construct (lane2) had the same size band as the positive control. Data
recorded on UTSA TVDC notebook #2, page108 for figure13.
3.6.7 The plasmid pKEK1149 (SIINFEKL) and pKEK1145(SIINFEKL) #1 were sent out for
sequencing with “PepO For” primer, and the sequencing result confirmed SIINFEKL insertion in
pKEK1145 and in pKEK1149. The plasmid pKEK1145 with SIINFEKL was named as
pKEK1169 and pKEK1149 with SIINFEKL and the Ft promoter was named as pKEK1168.
The sequencing data recorded on UTSA TVDC notebook #2, page116.
3.6.8 Western blotting was used to show the expression of the SIINFEKL peptide, associated
with two different plasmids. The whole cells lysates were made for the West Blotting to detect
the expression of SIINFEKL from pKEK1168 and pKEK1169 in E.Coli.. For pKEK1168,
overnight culture was used, and as for pKEK1169, 1.5 Arabinose was added to the overnight
culture and 4 hours culture to induce the protein expression since pKEK1169 was a PBAD
plasmid. PBAD plasmid contained Arabinose dependent PBAD promoter (in the plasmid 12501277bp). With Arabinose in the culture, it would induce the transcription of the gene cloned into
this plasmid. For the Western blotting, the primary antibody was anti-SIINFEKL from cells
culture and the second antibody was anti-mouse antibody. PepO-SIINFEKL protein was about
78KD.
19 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 20 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Figure 14: The picture of the Western Blotting for detection of SIINFEKL expression.
1
2
3
4
5
1.Pkek1169 overnight culture
2.pKEK1168 overnight culture
3.OVA
4.Pkek1169 4hrs culture
5.DH5 E.coli.
75K
D
50K
D
Figure 14 title, legend and data location: The Western Blotting for detection of SIINFEKL expression from
pKEK1168 and pKEK1169 in E.Coli. Lane5 (E.coli) was the negative control, and lane3 was supposed to be the
positive control. Lane1 was overnight culture of pKEK1169 with the expression of SIINFEKL. Lane4 was 4 hours
culture of pKEK1169 with SIINFEKL being expressed. Lane2 was overnight culture of pKEK1168 without any
expression from SIINFEKL. Data are located in TVDC UTSA Notebook#2, page 110
There was no SIINFEKL expression from pKEK1168, but the corresponding band to the
SIINFEKL protein of approximately 75KD was shown from pKEK1169 in the overnight culture
and the 4 hr culture. No positive signal from OVA was possibly because the anti-SIINFEKL
didn’t recognize the antigen site in OVA.
3.6.8 To confirm the SIINFEKL expression in E.Coli. from pKEK1169 by the Western Blotting,
the same procedure was used, and a kinetic time course was set up. 1.5 Glucose was added in
overnight culture to inhibit the protein expression (lane1), and 1.5 Arabinose was added in
separate cultures at time point 1hr, 2hrs, 3hrs, 4hrs and overnight (lane2-6) to induce protein
expression.
Figure 15: The picture of the Western Blotting for detection of SIINFEKL expression under the
time course.
1
2
3
4
5
6
7
75KD
50kd
1.Overnight culture forpkek1169/
2.1hr culture for pkek1169
3.2hrs culture for pkek1169
4.3hrs culture for pkek1169
5.4hrs culture for pkek1169
6.Overnight culture forpkek1169
7.DH5  E.coli.
Lane1: with1.5 glucose
Lane2-6: with 1.5 Arabinose
Figure 15 title, legend and data location: The Western Blotting for detection of SIINFEKL expression from
pKEK1169 in E.Coli. Lane7 (E.coli) was the negative control. Lane1 was the overnight culture with 1.5% Sucrose
which inhibited the expression of protein. Lane2-lane6 were 1hr, 2hrs, 3hrs, 4hrs, and overnight cultures with 1.5%
Arabinose in each culture to induce the expression of protein. The result confirmed that there was SIINFEKL
expression from pKEK1169 in E.Coli. since the SIINFEKL protein expression was increasing as the time went on.
Data are recorded on UTSA TVDC notebook #2, page 111 for Figure15.
20 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 21 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
3.6.9 Since pKEK1168 (with Francisella promoter) did not express SIINFEKL in DH5 E.coli., a
new plasmid with both PepO-SIINFEKL and Francisella promoter was created. Both the pBAD
plasmid pKEK1169 with PepO-SIINFEKL and pKEK894 with F. promoter were double digested
with NcoI and PstI, then PepO- SIINFEKL released from pKEK1169 was ligated into the
backbone pKEK894 and transformed into DH5 E.coli. The transformants were screened by
colony PCR.
3.6.10 The primers used for colony PCR to confirm PepO-SIINFEKL insertion in pKEK894
were:
pFn Bgl2 for (in F. promoter seq.): 5’ ggg aga tct ttt ggg ttg tca ctc atc gta ttt g
SIINFEKL Rev (in SIINFEKL seq.): 5’ cgc gct gca gct aaa gct ttt cga aat
3.6.11 Set up following colony PCR to confirm the insertion:
dd water
36.5ul
10x XL buffer
5.0ul
KOD dNTPs
5.0ul
DNA Template
1.0ul
SIINFEKL Rev
1.0ul
pFn Bgl2 for
1.0ul
XL DNA polymerase
0.5ul
At 94C 2min, then 94C 30 sec/55C 30sec/ 72C 2min30sec// 30 cycles, then 72C 10 min
Figure 16: The gel picture of colony PCR to screen pKEK894/PepO-SIINFEKL transformants.
1
2
3
4
5
6
7
8
Lane1 1kb Ladder
Lane2 pKEK1168
Lane3 pKEK894
Lane4 colony1
Lane5 colony2
Lane6 colony3
Lane7 pKEK1169
Lane8 1kb Ladder
3.0Kb
2.1kb
1.5kb
Lane4-lane6 pKEK894 with PepO-SIINFEKL
21 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 22 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Figure 16 title, legend and data location: Colony PCR for confirmation of PepO-SIINFEKL in pKEK894. Lane2
and 7 were the positive controls, and lane3 was the negative control. Lane4-6 (colony1-3) showed the same size
band as the positive controls (about 2.0kb). Data are located in TVDC UTSA Notebook#2, pages 113.
The gel purified PCR DNA was sent for sequencing with “PepO For “ and “pFnBglII for” for
primers and the result confirmed the insertion. The new plasmid was named as pKEK1177
containing PepO-SIINFEKL under the control of only the Francisella promoter from the
backbone pKEK894.
The sequencing data recorded on UTSA TVDC notebook #2, page117.
3.6.12 The Western Blotting was performed to detect the SIINFEKL expression from pKEK1177
in DH5 E.coli. with the whole cells lysates from overnight culture. The primary antibody was a
mouse monoclonal antibody to SIINFEKL and the second antibody was anti-mouse antibody.
Figure 17: The picture of the Western Blotting for detection of SIINFEKL expression
1
2
3
4
75KD
1.pKEK1169
2.DH5α
3.pKEK1177, colony1
4.pKEK1177, colony2
Figure 17 title, legend and data location: The Western Blotting for detection of SIINFEKL expression from
pKEK1177 in E.Coli. Lane1(pKEK1169) was the positive control, and lane2 (E.coli) was the negative control.
Lane3 and 4 (pKEK1177) presented the expression of SIINFEKL in E.coli, but the signal was weak compared to
lane1. Data is located in TVDC UTSA Notebook#2, pages -113.
The plasmid pKEK1177 expressed SIINFEKL in E.Coli., but the signal was weak relative to
pKEK1169
22 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 23 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
3.6.13 The plasmid pKEK1177 was transformed into both wild type LVS and F. novicida U112 using
electroporation. The transformants were selected on 10ug/ml Tetracycline TSA++ plate.
3.6.14 Screened the colonies by colony PCR with “SIINFEKL Rev “and “ pFnBglII For “ primers.
PCR reaction was set up as step 4.9.
Figure 18: The gel picture of colony PCR for pKEK1177 in LVS and U112.
Figure 18 title, legend and data location: Colony PCR for confirmation of PepO-SIINFEKL in LVS and U112.
Lane2 (pKEK1177) was the positive control, and lane3 (wt U112) and lane4 (wt LVS) were the negative controls.
Lane5-lane7 were LVS/pKEK1177 #1-3, which presented the same pattern of PCR as lane2. Lane8-10 were
U112/pKEK1177 #1-3, which also showed the same size of bands as the positive control. Data is located in TVDC
UTSA Notebook#2, page114.
PCR products about 2.2kb from lane5 to lane10 with the positive control on lane2 and negative
controls on lane3 and lane4 confirmed that the pKEK1177 had been transformed into both wild type
LVS ( and U112 successfully.
3.6.15 Performed western blotting for LVS with pKEK1177 and U112 with pKEK1177 using the
whole cells lysates from overnight culture to detect the expression of SIINFEKL. The primary
antibody was monoclonal anti-SIINFEKL from the University of Massachusetts Medical School and
the second antibody was anti-mouse antibody purchased from GE Healthcare. PepO-SIINFEKL
protein was about 78KD (Data shown in Figure 19).
23 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 24 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Figure 19: The picture of the Western Blotting for SIINFEKL expression from LVS/pKEK1177
and U112/pKEK1177.
Figure 19 title, legend and data location: The Western Blotting for detection of SIINFEKL expression in LVS and
U112. Three colonies (lane2 to lane4) from LVS with pKEK1177 and 2 colonies (lane7 and lane8) from U112 with
pKEK1177 expressed SIINFEKL. Lane1 and lane5 were the negative controls. The created T-cell epitope tagged
PepO-SIINFEKL was expressed by F. tularensis, in both LVS and U112 F novicida. Data are located in TVDC
UTSA Notebook#2, pages 114.
3.6.16 LVS with pKEK1177 was named as “KKF320” and U112 with pKEK1177 as “KKF319”.
KKF320 and KKF319 are additional deliverables on Milestone 43
Data recorded on UTSA TVDC notebook #2, page112-114 for figure16-19.
24 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 25 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Critical reagents:
XL DNA Polymerase and KOD HiFi DNA Polymerase (Novagen)
Kanamycin and Chloramphenical Antibiotics (Sigma)
Bgl2 Restriction Enzyme (NEB)
Anti-SIINFEKL Antibody (the University of Massachusetts Medical School)
Anti-mouse Antibody (GE Healthcare)
SOP Number1
UTSA1
UTSA2
UTSA4
SOP Title
Generation of Electrocompetant E.coli and transformation
procedure
Generation of Electrocompetant Francisella and Transformation
Procedure
Conjugation of the plasmid into Francisella tularensis LVS
1
Individual Standard Operating Procedures will be reviewed separately and accepted by the Subcontracting PI and UNM
PI.
4
Salient Original Data, Results, Interpretation, Quality Control
4.1
Original Data and Results (Rationale, Tables/Figures with legends and location
annotations)
The original data and results are intermixed with the Methods in section 3 above. Combining
the methods and results improved the flow of the MS43 MSCR.
4.2
Interpretation
4.2.1 The uvrA and uvrB mutants of LVS have been generated.
4.2.2 LVS with PepO-SIINFEKL and F. novicida U112 with PepO-SIINFEKL have been
created.
4.3
Quality Control
Mutants KKF303 and KKF317, mutant constructs pKEK1168, pKEK1169 and pKEK1177
have been confirmed by DNA sequencing.
Mutants KKF319 and KKF320 have been confirmed by PCR. The expression of SIINFEKL
in both KKF319 and KKF320 was confirmed by the Western Blotting.
Mutant construct pKEK1167 was confirmed by digestion with Bgl2.
Mutant constructs pKEK1028, pKEK1029 and pKEK1090 were confirmed by PCR.
25 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Version: 1.0
Page 26 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
5
Accepted
Date:10/22/08
Deliverables Completed
Delivered LVS uvrA and uvrB to UNM and Cerus.
Delivered LVS with PepO-SIINFEKL and F.novicida U112 with PepO-SIINFEKL to UNM and
Cerus.
Created plasmids required for the production of the uvrA and uvrB mutants of LVS, the LVS
pepO-SIINFEKL and the Fnovicida U112 with PepO SIINFEKL:
pKEK1028(uvrBLVS up), pKEK1029(uvrBLVSDn); pKEK1090(GroELp
plasmid),pKEK1114(uvrBFpKan plasmid), KKF303(uvrB mutant LVS); pKEK1167(Targetron
plasmid), KKF317(uvrA mutant LVS); pKEK1168(Fp-PepO-SIINFEKL plasmid),
pKEK1169(PepO-SIINFEKL plasmid), pKEK1177(Fp–PepO-SIINFEKL plasmid),
KKF319(PepO-SIINFEKL U112), KKF320(PepO-SIINFEKL LVS).
NOTE: The assessment of the Ft novicida mutants in a KBMA vaccine platform,
would have been performed by Cerus under Cerus' MS#43; however, Cerus’ Milestone #43 was
terminated.
Under Milestone #43, Cerus will not assess the Ft novicida mutants, generated by UTSA.
Cerus' statement of work changed and the rationale is included in the
modified Cerus subcontract (COA 4R2). In brief, "since the mechanism of
immunity against Ft novicida is humoral and heat killed vaccines perform
as well as KBMA, thus Cerus cannot compare the potency of various KBMA
platform strains" for Ft novicida.
Deliverable Reagents
Bacterial
Strain
KKF303
# of
vials
Vial
Concentra
tion
(uvrA mutant in
LVS)
Date Stored
or Date
Transferred*
Storage location*
UTSA, BSE 3.242 ,
1.Primary -80ºc freezer,
Box “KKF244-”;
2.Backup -80ºc freezer,
Box “KKF244- backup”
UTSA, , BSE 3.242 ,
1.Primary -80ºc freezer,
Box “KKF244-”;
2.Backup -80ºc freezer,
Box “KKF244- backup”
2
109 cfu/ml
Glycerol
stock
03/23/2007
2
109 cfu/ml
Glycerol
stock
07/27/2007
(uvrB mutant in
LVS)
KKF317
Storage
media
(Institution, room, shelf, etc)
26 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Version: 1.0
Page 27 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
KKF319
2
109 cfu/ml
Glycerol
stock
08/28/2007
2
109 cfu/ml
Glycerol
stock
08/29/2007
4
109 cfu/ml
Glycerol
stock
11/06/2007
4
109 cfu/ml
Glycerol
stock
11/06/2007
4
109 cfu/ml
Glycerol
stock
11/06/2007
4
109 cfu/ml
Glycerol
stock
11/06/2007
2
109 cfu/ml
Stab
06/20/2007
UNM BRF G72, dedicated
storage box for the
mutants in -80ºc
freezerB8,9;C1
UNM BRF G72, dedicated
storage box for the
mutants in -80ºc freezer;
C2-4
Cerus
2
109 cfu/ml
Stab
09/05/2007
Cerus
2
109 cfu/ml
Stab
09/05/2007
Cerus
2
109 cfu/ml
Stab
09/05/2007
Cerus
(PepOSIINFEKL
U112)
KKF320
(PepOSIINFEKL
LVS)
KKF303
(uvrB mutant in
LVS)
KKF317
(uvrA mutant in
LVS)
KKF319
(PepOSIINFEKL
U112)
KKF320
(PepOSIINFEKL
LVS)
KKF303
Accepted
Date:10/22/08
UTSA, , BSE 3.242 ,
1.Primary -80ºc freezer,
Box “KKF244-”;
2.Backup -80ºc freezer,
Box “KKF244- backup”
UTSA, , BSE 3.242 ,
1.Primary -80ºc freezer,
Box “KKF244-”;
2.Backup -80ºc freezer,
Box “KKF244- backup”
UNM BRF G72, dedicated
storage box for the
mutants in -80ºc freezer ;
B2-4
UNM BRF G72, dedicated
storage box for the
mutants in -80ºc freezer,
B5-7
(uvrB mutant in
LVS)
KKF317
(uvrA mutant in
LVS)
KKF319
(PepOSIINFEKL
U112)
KKF320
(PepOSIINFEKL
LVS)
27 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Version: 1.0
Page 28 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Tissue
identifier
# of
blocks
RNA/DNA/
Plasmids
pKEK1028
# of
vials
Tissue
Type
Vial
Concentra
-tion
~ Mass
per
block
Storage
media
Date Stored
or Date
Transferred*
Storage location*
Date Stored
or Date
Transferred*
Storage location* and
**(Institution, room, shelf, etc)
UTSA, BSE 3.242 ,
1.Primary -80ºc freezer,
Box “KEK1028-”;
2.Backup -80ºc freezer,
Box “KEK1028- backup”
UTSA, , BSE 3.242 ,
1.Primary -80ºc freezer,
Box “KEK1028-”;
2.Backup -80ºc freezer,
Box “KEK1028- backup”
UTSA, , BSE 3.242 ,
1.Primary -80ºc freezer,
Box “KEK1028-”;
2.Backup -80ºc freezer,
Box “KEK1028- backup”
UTSA, , BSE 3.242 ,
1.Primary -80ºc freezer,
Box “KEK1109-”;
2. Backup -80ºc freezer,
Box “KEK1109- backup”
UTSA, BSE 3.242 ,
1.Primary -80ºc freezer,
Box “KEK1109-”;
2.Backup -80ºc freezer,
Box “KEK1109- backup”
UTSA, BSE 3.242 ,
1.Primary -80ºc freezer,
Box “KEK1109-”;
2. Backup -80ºc freezer,
Box “KEK1109- backup”
UTSA BSE 3.242 ,
2
109 cfu/ml
In Ecoli
07/01/2006
2
109 cfu/ml
In Ecoli
07/01/2006
2
109 cfu/ml
In Ecoli
10/02/2006
2
109 cfu/ml
In Ecoli
11/06/2006
2
109 cfu/ml
In Ecoli
05/18/2007
2
109 cfu/ml
In Ecoli
05/24/2007
2
109 cfu/ml
In Ecoli
05/25/2007
(uvrBLVS up)
pKEK1029
(uvrBLVSDn)
pKEK1090
(GroELp)
pKEK1114
(uvrBFpKan)
pKEK1167
(Targetron)
pKEK1168
(Fp-PepOSIINFEKL)
pKEK1169
Accepted
Date:10/22/08
(Institution, room, shelf, etc)
28 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Version: 1.0
Page 29 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
(PepOSIINFEKL)
pKEK1177
109 cfu/ml
2
In Ecoli
08/08/2007
(Fp–PepOSIINFEKL)
Polypeptide
#
Concentra
-tion
Storage
media
Date Stored
or Date
Transferred*
Accepted
Date:10/22/08
1.Primary -80ºc freezer,
Box “KEK1109-”;
2.Backup -80ºc freezer,
Box “KEK1109- backup”
UTSA, BSE 3.242 ,
1.Primary -80ºc freezer,
Box “KEK1109-”;
2.Backup -80ºc freezer,
Box “KEK1109- backup”
Storage location*
(Institution, room, shelf, etc)
*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.
** Barbara asked Rick Lyons if UNM wants any of the plasmids that UTSA used to make the uvrA, uvrB or pep O LVS,
novicida. Rick replied that UTSA can maintain the repository of the plasmids and UNM wants the 4 vials of each of the
bacterial mutants (KKF320, KKF319, KKF317 and KKF303). Rick does not want UNM to be repository for the cadre of
plasmids needed to make the appropriate LVS and F novicida strains. 9/23/08
6
Appendices
6.1
Appendix 1: Original Data Tables and Figures
Table/
Figure1
F-1
Title
Notebook Location2
(Notebook # and page
numbers)
Amplification of UvrB gene from the
potential UvrB mutant LVS chromosomal
DNA
TVDC UTSA
Notebook#2, page 67
Electronic
Location2 (Full
Path & File Name)
UTSA Asset
151425/C:/Ping/Figu
res for MS43/
20070326 uvrb
pcr(0320) to be
cut.jpg
F-2
Amplification of SacB gene from the
TVDC UTSA
UTSA Asset
29 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Version: 1.0
Page 30 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Table/
Figure1
Title
Accepted
Date:10/22/08
Notebook Location2
(Notebook # and page
numbers)
potential UvrB mutant LVS chromosomal
DNA
Notebook#2, page 68
Electronic
Location2 (Full
Path & File Name)
151425/C:/Ping/Figu
res for MS43/ F-2
ms43.jpg
F-3
Digestion of mutant UvrB gene from
UvrB mutant LVS with Bgl2
TVDC UTSA
Notebook#2, page 67
UTSA Asset
151425/C:/Ping/Figu
res for MS43/
20070322 uvrb
pcr dna cut with
bgl2.jpg
F-4
Amplification of Kanamycin selection
marker gene from the potential UvrA
mutant LVS colony
TVDC UTSA
Notebook#2, page 74
UTSA Asset
151425/C:/Ping/Figu
res for MS43/
20070427 uvrAKan
LVS colony
pcr.jpg
F-5
Amplification of 350bp PCR product for
re-targeting the intron RNA
TVDC UTSA
Notebook#2, page 76
UTSA Asset
151425/C:/Ping/Figu
res for MS43/
20070501350bp
pcr.jpg
F-6
Digestion of the potential Tulatron vector
DNA with Bgl2
TVDC UTSA
Notebook#2, page78
UTSA Asset
151425/C:/Ping/Figu
res for MS43/
20070517 uvrA
mini prep cut
WITH BglII.3.jpg
F-7
Colony PCR for confirmation of the
intron insertion in UvrA gene of LVS
TVDC UTSA
Notebook#2, page80
UTSA Asset
151425/C:/Ping/Figu
res for MS43/
20070625 pcr
with uvrAschu4up
and universal
primers.jpg
F-8
Colony PCR for confirmation of the
intron insertion in UvrA gene of LVS
TVDC UTSA
Notebook#2, page81
UTSA Asset
151425/C:/Ping/Figu
res for MS43/ uvra
lvs with
uvraschu4lvsdn
and
uvraschu4up.jpg
30 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Version: 1.0
Page 31 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Table/
Figure1
F-9
Title
Accepted
Date:10/22/08
Notebook Location2
(Notebook # and page
numbers)
Colony PCR for confirmation of the
UvrA mutant LVS with the plasmid
being removed
TVDC UTSA
Notebook#2, page 83
Electronic
Location2 (Full
Path & File Name)
UTSA Asset
151425/C:/Ping/Figu
res for MS43/ EBS
universal,
urraschu4up and
uvrAshcu4lvsDn.2
.jpg
F-10
Amplification of SIINFEKL fragment
TVDC UTSA
Notebook#2, page104
UTSA Asset
151425/C:/Ping/Figu
res for MS43/
20070522
SIINFEKL.jpg
F-11
The miniprep DNA of the transformant
pKEK1145 with SIINFEKL
TVDC UTSA
Notebook#2, page106
UTSA Asset
151425/C:/Ping/Figu
res for MS43/
20070525 mini
prep for
pKEK1145
SIINFEKL.jpg
F-12
Colony PCR for confirmation of
SIINFEKL in pKEK1145
TVDC UTSA
Notebook#2, page107
UTSA Asset
151425/C:/Ping/Figu
res for MS43/
20070531 pcr for
SIINFEKL
insertion,
pGEM-T mini
prep.2.jpg
F-13
PCR for confirmation of SIINFEKL in
pKEK1149
TVDC UTSA
Notebook#2, page108
UTSA Asset
151425/C:/Ping/Figu
res for MS43/
pKEK1149SIINFEKL with
PepO For and
SIINFEKL Rev.jpg
F-14
The Western Blotting for detection of
SIINFEKL expression from pKEK1168
and pKEK1169 in E.Coli.
TVDC UTSA
Notebook#2, page110
UTSA Asset
151425/C:/Ping/Figu
res for MS43/
Western
figure1.ppt
F-15
The Western Blotting for detection of
SIINFEKL expression from pKEK1169
TVDC UTSA
UTSA Asset
151425/C:/Ping/Figu
31 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Version: 1.0
Page 32 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Table/
Figure1
Title
Accepted
Date:10/22/08
Notebook Location2
(Notebook # and page
numbers)
in E.Coli.
Notebook#2, page 111
Electronic
Location2 (Full
Path & File Name)
res for MS43/
WESTERN FIGURE 2
.ppt
F-16
Colony PCR for confirmation of PepOSIINFEKL in pKEK894
TVDC UTSA
Notebook#2, page113
UTSA Asset
151425/C:/Ping/Figu
res for MS43/
figure for
pKEK1177 colony
PCR.jpg
F-17
The Western Blotting for detection of
SIINFEKL expression from pKEK1177
in E.Coli.
TVDC UTSA
Notebook#2, page113
UTSA Asset
151425/C:/Ping/Figu
res for MS43/
western
figure3.ppt
F-18
Colony PCR for confirmation of PepOSIINFEKL in LVS and U112
TVDC UTSA
Notebook#2, page114
UTSA Asset
151425/C:/Ping/Figu
res for MS43/
kek894(peposiinfekl) lvs
and u112 with
SIINFEKL Rev and
pFnBglII for.jpg
F-19
The Western Blotting for detection of
SIINFEKL expression in LVS and U112
TVDC UTSA
Notebook#2, page 114
UTSA Asset
151425/C:/Ping/Figu
res for MS43/
western
figure4.JPG
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
technical report, then provide both the previously reported data location and the final data location
1
2
32 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Version: 1.0
Page 33 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
6.2
Accepted
Date:10/22/08
Appendix 2: Quality Assessment of Milestone Completion and Report
Assessment Criteria for Milestone Completion
Evaluation of Milestone
Completion Report
Yes
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?
X
Well done! Ping listed the data
locations for the DNA
sequencing files in appendix
6.3.1 on 9/30/08
X
Edited version accepted
9/30/08
X
SOP 1 and 2 were submitted to
NIAID in association with
another MSCR from UTSA.
SOP4 ver. 0.2 is accepted by
Dr. Klose and will be
submitted to NIAID in
conjunction with MS 43
MSCR.
Are salient negative and positive original data
included in the Milestone Completion Report?
Has the Deliverables Table been completed?
X
X
Have the Appendices been completed?
X
Are the specific original data associations
with experiments clearly annotated in the
“Salient Technical Data” section of the
Milestone Completion report?
X
Evaluation of Data included
Yes
Are the salient original data and results
included in an organized, easily interpretable
format?
Is the rationale included?
X
Do tables and figures have legends and
original data location annotations?
Is the data interpretation clear?
X
X
No N/A
Comment
location edits completed and
received all needed vials on
10/16/08 at UNM
sequencing data locations were
added to appendix 6.3.1
9/30/08
Actually methods and salient
data are combined under
methods section.
No N/A
Comment
edits for simple text transitions
between strategies added
9/30/08 Data is fine.
completed in ver. 0.3 on
9/30/08
X
33 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 34 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Assessment Criteria for Milestone Completion
Is the data storage location listed in Appendix
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?
X
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?
X
X
DNA Sequencing, PCR,
western blotting have provided
good quality control
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
Proteins have been confirmed
by Western blotting.
If a genetic mutant has been made, how has
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?
X
DNA sequencing, PCR
verification, number of
mutants examined.
Hardcopy and some electronic
too
UTSA has a fireproof cabinet
for notebooks
X
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?
X
34 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 35 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Assessment Criteria for Milestone Completion
Evaluation of Deliverables, as
outlined in the Statement of Work
Yes
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,
revised by subcontractor as requested, and
accepted by UNM?
X
Has data from the milestone been submitted
by the subcontractor, reviewed by UNM, data
presentation revised by the subcontractor as
requested for clarity, and accepted by UNM?
X
For deliverable reagents, have the minimum
number of vials, the minimum concentration,
the minimum block size and the minimum
weight of tissue been mutually agreed by
UNM and the subcontractor?
X
Have bacterial strains and tissues been 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.
X
X
No N/A
Comment
Yes, UNM has accepted SOP
1,2, and 4; NIAID still needs to
review SOP 4 ver. 0.2. which
Dr. Klose has accepted and
will be sent to NIAID with
MS#43 MSCR.
IT will be accepted when
UTSA sends the additional
final bacterial vials to UNM.
UTSA provided MS 43 MSCR
on approx 2/26/08. BG
reviewed it on 9/23/08 and
apologizes for the delay. UNM
accepted on 10/22/08 after
vials were rec’d on 10/16/08
UTSA provided better figure
legends for each of the 19
figures on 9/30/08 in ver. 0.3
of the MS#43 MSCR So this is
done.
For each deliverable LVS and
Fn strain, UNM needs 4 vials,
so 2 can be retained at UNM
long term and 2 can be
transferred to NIAID at the end
of the contract. Rick does
NOT want UNM to be a
repository for the various
plasmid constructs leading up
to the LVS and Fnovicida
mutant bacteria 9/23/08. Vials
were rec’d 10/16/08 so this is
done.
On 10/16/08, vials were
received at UNM so the MSCR
is completed.
35 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Accepted
Date:10/22/08
Version: 1.0
Page 36 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
Assessment Criteria for Milestone Completion
Evaluation of SOPs
Yes
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 can read and understand the standard
operating procedure?
X
No N/A
Comment
Well written! An independent
scientist can read and
understand the standard
operating procedure?
SOP#4 ver. 1.0 accepted by
NIAID
36 of 37
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 # 43
Institution: UTSA
Author: PING CHU/ JIRONG LIU
MS Start
Date:05/01/2006
MS End Date:
08/31/2007
Report Date:
02/25/2008
Version: 1.0
Page 37 of 37
Reviewed by : Barbara Griffith 09/23/08, 9/30/08,
10/22/08, 11/04/08, 11/06/08
6.3
Accepted
Date:10/22/08
Appendix 3: Additional Data/Figures not included in the Text of the Milestone
Completion Report (Section 4)
6.3.1 Mutants KKF303 and KKF317, mutant constructs pKEK1168, pKEK1169 and
pKEK1177 have been confirmed by DNA sequencing.
Mutants
Mutant Constructs
Electronic File Location of Sequencing Data
UTSA Asset 151425/C:/Ping/Sequencing for
MS43/ Kan6_KanFNdel_C04.seq
KKF303
UTSA Asset 151425/C:/Ping/Sequencing for
MS43/ Kan6_KanRBam1-1I_D04.seq
UTSA Asset 151425/C:/Ping/Sequencing for
MS43/ UvrALus2_EBSUniversal_D03.seq
UTSA Asset 151425/C:/Ping/Sequencing for
MS43/ UvrALus2_UvrASchu4Up_C03.seq
KKF317
UTSA Asset 151425/C:/Ping/Sequencing for
MS43/ UvrALus4_EBSUniversal_B03.seq
UTSA Asset 151425/C:/Ping/Sequencing for
MS43/ UvrALus4_UvrASchu4Up_A03.seq
pKEK1168
UTSA Asset 151425/C:/Ping/Sequencing for
MS43/ 1149SIINFEKL_PepoFor_H05.seq
pKEK1169
UTSA Asset 151425/C:/Ping/Sequencing for
MS43/ 1145SIINFKL1_PepoFor_G05.seq
UTSA Asset 151425/C:/Ping/Sequencing for
MS43/ pKEK894SIIN_PepoFor_D02_seq
pKEK1177
UTSA Asset 151425/C:/Ping/Sequencing for
MS43/ pKEK894SIIN_pFnBglIIfor_E02_seq
37 of 37