UNM TVDC UTSA - UNM Tech Call Minutes: 2/20/2007
Prepared 2/20/2007: Mindy Tyson/Barbara Griffith
Sent to UTSA for review: 2/20/07
Reviewed: Karl Klose 2/23/07 and Bernard Arulanandam 2/21/07
Distributed to NIAID on: 2/23/07
Present: Karl Klose, Barbara Griffith, Vicki Pierson, Marlene Hammer, Bernard Arulanandam,
Rick Lyons
Absent: Joe Breen, Kristin DeBord
Action Items from 2/20/07 Call:
 Karl will share the new slides about a day before the presentation during the week of 2/26.
 Barbara will send Rick’s old slides to Karl from the kickoff TVDC meeting. (completed
2/20/07)
Residual January action items:
 Barbara has milestone completion reports for MS 16 &39 from UTSA and will be reviewingsent comments on MS#16 report to UTSA on 2/1/07- Karl: please respond to Barbara’s
comments on MS#16 Milestone completion report.
 Karl will ask Jeff Barker to provide UNM the MS 48 completion report (gave to Jeff 1/23/07)Barbara has not yet received this from Karl or Jeff as of 2/20/07
The meeting was recorded for the purposes of the minutes.
A. Active milestones during last reporting period:
a. Milestone #43: Creation of uvrA and uvrB mutant F. tularensis subsp. holarctica
(LVS) strains
b. Milestone #49A: Construction of iglC F. tularensis subsp. tularensis strain (SCHU
S4)
c. Milestone #50A: Immunologic characterization of F. tularensis subsp. novicida,
subsp. tularensis, and LVS strains
d. Milestone #51: Construction of F. tularensis subsp. Novicida uvrA + pdpD, iglA,
iglB, iglC, iglD and uvrB + pdpD, iglA, iglB, iglC, iglD strains.
B. Milestone #43 Flow Chart: Creation of uvrA and uvrB mutant F. tularensis subsp.
holarctica (LVS) strains
a. Creation of uvrA and uvrB mutant F. tularensis subsp. holarctica (LVS) strains
1. Construct uvrB::Kan mutagenesis plasmid
a. Mate into LVS
b. Select for transconjugate
c. Counterselect for mutant
2. Construct uvrA::Kan mutagenesis plasmid
a. Mate into LVS
b. Select for transconjugate,
c. Counterselect for mutant
b. Verify mutants,
c. Pass on to Milestone 50
C. Milestone #43: Creation of uvrA and uvrB mutant F. tularensis subsp. holarctica
(LVS) strains
a. uvrB::Kan in mutagenesis plasmid mated with LVS; went in successfully
b. LVS strain with uvrB::Kan in chromosome, but this strain has not yet lost
plasmid backbone
c.
d.
e.
f.
g.
h.
i.
j.
Sucrose counterselection didn’t work, so we attempted to remove NaCl from
media to repeat this step (this generally facilitates sucrose efficacy)
The TSA media components needed to be ordered separately in order to remove
NaCl, these are on back order and still haven’t arrived.
However, we tried supplementing LB with enrichment components (“++”: Na
pyruvate, Na bisulfite, Fe sulfate, casamino acids and cysteine) that facilitate
growth of LVS on TSB, since we can leave NaCl out of LB.
LVS failed to grow on LB ++ media (documented in UTSA TVD notebook #2)
Karl has hired a replacement for Jirong Liu and the new person begins on
2/21/07
Rick: what is the back up plan if UTSA can’t remove the plasmid backbone?
Karl: UTSA knows that targetron works in SCHU S4 and novicida and expect
would also work in LVS.
Karl: will try other media that LVS will grow on, and will add sucrose to
Chamberlain, Mueller Hinton and another media. UTSA is halfway along on this
construct and anticipates that another media with sucrose will facilitate removal
of the plasmid backbone.
D. Milestone 49 Flow Chart: Creation of mutant F. tularensis subsp. tularensis strains
a. Construct iglC mutagenesis plasmid(s)- currently in progress.
1. Transform into Schuh4,
2. Select for transconjugate
3. Counterselect for mutant
b. Construct pdpD, iglD mutagenesis plasmids
1. Mate into Schuh4,
2. Select for transconjugate
3. Counterselect for mutant
c. Construct iglA, iglB mutagenesis plasmids
1. Mate into Schuh4,
2. Select for transconjugate
3. Counterselect for mutant
b. Verify mutants
c. Pass on to Milestone 50
E. Milestone #49A: Construction of iglC F. tularensis subsp. tularensis strain
a. Several concurrent strategies:
1. Strategy 1: create plasmid similar to published (colE1 ori)
a. We created sacB plasmid last period,we have now
cloned mglA deletion into this plasmid with 1 kbp
flanking homology.
b. We utilize mglA as our test mutation to develop these
technologies, because there is only one chrom. copy
(i.e.only one gene to knockout), and there is easy
phenotype associated with it (Acp-, gives white colony
on X-P media) Blue on other media.
c. mglA deletion appears to be stable (no deletions, unlike
previous experience with iglC deletion)
d. Will transform into Schuh4 this period
e. MglA is for proof of principal and then will work with iglC.
f. Oligo ordered to amplify iglC deletion to place in this
plasmid
g. Hard to get the highcopy number iglC plasmid into
SCHU S4.
h. Rick: Is UTSA having trouble getting iglC gene into the
high copy number plasmid?
i.
Karl: yes, that is the problem, most likely. High copy
number is unlikely to work but UTSA will keep trying it.
j. Rick: is the high copy number plasmid a toxic effect?
k. Karl: yes, likely. iglC with a little bit of expression, then
can’t clone it, especially when IglD is associated.
2. Strategy 2: use new mating plasmid (oriR6K)
a. We performed ligation of iglC into mating plasmid
pKEK1090, evaluated 15 resultant colonies, none had
insert.
b. UTSA will repeat, and screen pools of hundreds of
colonies to identify correct clone.
3. Strategy 3: (Value added) Targetron system
a. We have adapted Targetron (intron targeting system of
Sigma) to work in Ft successfully.
b. We have created new Targetron plasmid that is
temperature-sensitive in Ft, this is critical for removing
plasmid after mutation introduced. Final strain should not
carry the plasmid. Will use temperature to remove
c. Rick: is it an inducible system?
d. Karl: no, it is on all the time. The plasmid has both
promoters, one for targetron and one for other gene.
Maybe 2-3 colonies initially have the targeted gene
mutations and the next round of rich media results in
maybe 10x more colonies with the targeted gene.
Targetron inserts an intron into a very specific site in the
gene being targeted and the gene remains but is
mutagenized by the insertion of the intron fragment and
the targeted gene therefore is not functional. If you
design the target carefully, targetron will work on all 3
species of Ft. Targetron may revolutionize making
mutants in all 3 strains. The gene mutation due to the
targetron intron insertion is permanently stable and Ft
doesn’t carry the enzyme to remove the intron. It is like
a transposon without the transposase so the intron can’t
move out.
e. We have designed oligos to knockout iglC, PCR
fragment has been amplified, we are in process of
screening Targetron plasmid with iglC insert
(documented in UTSATVD Notebook 3).
f. Targetron targets the gene of interest. Every gene tried
in novicida and SCHU S4 has worked to date. It keeps
targeting the gene until there are no copies of the
original gene left. It will inactivate both copies of the
genes.
F. Milestone 51 Flow Chart: Creation of uvrB + pdpD, iglA, iglB, iglC, and iglD mutant
F. tularensis subsp. novicida double mutant strains
a. Construct uvrB::Kan mutagenesis plasmid
1. Transform into pdpD, iglA, iglB, iglC, iglD novicida strains, isolate
mutant
a. Verify mutants,
b. Pass on to Milestone 50
b. Construct iglB::ermC mutagenesis plasmid
1. Transform into U112, isolate mutant
G. Milestone #51: Creation of uvrB + pdpD, iglA, iglB, iglC, iglD mutant F. tularensis
subsp. novicida strains
a. Upon consultation with Rick and Justin, we are constructing five strains, by
moving uvrB::Kan mutation into strains with single ermC insertions in pdpD, iglA,
iglB, iglC, and iglD
b. We now have all the single ermC mutant strains
c. Put a different marker in uvrB
d. uvrB::Kan chromosomal DNA prepared from Ft novicida uvrB::Kan strain,
cryotransformed into iglC::ermC strain, KanR colonies selected
e. Colonies screened, many colonies were correct and one colony verified to be
uvrB::Kan iglC::ermC double mutant, named KKF224 (documented in
UTSATVD Notebook 2).
H. Milestone 50 Flow Chart: Immunologic characterization of F. tularensis subsp.
novicida, subsp. tularensis, and LVS strains
a. F. novicida uvrA, uvrB Double mutant
1. In vitro Growth
2. In vivo Bacterial Burden
3. LD50 determination
b. F.novicida uvrB+pdpD iglA, iglB, iglC, iglD
1. In vitro Growth
2. In vivo Bacterial Burden
3. LD50 determination
a. Further immunological characterization based on initial
screen
c. LVS uvrA, uvrB F. tularensis Schu4 iglC
1. In vitro Growth
2. In vivo Bacterial Burden
3. LD50 determination
a. Further immunological characterization based on initial
screen
I.
Milestone #50A: Immunologic characterization of F. tularensis subsp. novicida,
subsp. tularensis, and LVS strains
a. Results Update
1. Determine the LD50 of Ft subsp. novicida iglB mutant
a. Groups of BALB/c mice (6 mice per group) were
challenged with ΔiglB (104-107 CFU per mouse via
intranasal (i.n.) route). Animals were monitored for
morbidity and mortality.
b. Figure 1:
i. Results: there is no mortality observed at any
dose, indicating the high degree of attenuation
with this organism. No significant weight loss of
infected mice was also observed through day 18
at any of the doses; is highly attenuated in mice.
No ruffles, no hunching etc. is observed in the
mice.
2. Monitor Ft subsp. novicida iglB mutant replication and
dissemination in mice after intranasal challenge
a. BALB/c mice were challenged with ΔiglB mutant (106
CFU) intranasally. Lungs, liver, spleen, and lymph nodes
were collected from the infected mice at a three day
interval (3 mice per time point) up to 15 days. Numbers
of bacteria in each organ were determined by dilution
plating.
b. Figure 2:
i. Results: As shown in Fig. 2, there was
heightened replication of the organism in the
lungs within the first 12 days, with reduction
noted at day 15. There were lower levels of
replication within the liver and spleen. Numbers
of bacteria in the spleen are consistent through
out the observed period. In the liver, the
bacterial burden was decreased by day 15.
There were organisms recovered from the
draining lymph nodes, but at much lower levels
than that seen with the other target organs.
ii. No physical signs of infection were observed
based on survival curves
iii. Question: Rick- wild type at 106, the animals
would be dead. What would be the organ
distribution?
iv. Rick: will the organs eventually clear the
infection with the attenuated strain?
v. Bernard: have watched up to day 30 and then
the bacteria is gone completely.
J.
Plan for following month:
a. Milestone #16: completed.
b. Milestone #39: completed.
c. Milestone #48: completed.
d. Milestone #43:
1. Re-perform counterselection to remove plasmid backbone from
LVS to create uvrB::Kan LVS mutant
e. Milestone #49A:
1. Clone iglC into colE1 mutagenesis vector – high copy vector
2. Clone iglC into R6K mutagenesis vector- low copy vector
3. Clone iglC targeting fragment into Targetron vector
f. Milestone #51:
1. Construct uvrB::Kan iglA::ermC double mutant
g. Milestone #50:
1. Evaluate the protective efficacy of the Ft subsp. novicida iglB
mutant as a vaccine candidate. Groups of vaccinated mice will
be challenged i.n. with lethal dose of Ft subsp. novicida.
Animals will be monitored for survival and weight loss.
2. Analyze the antibody profiles of mice immunized with the Ft
novicida iglB mutant at day 30 after vaccination.
K. Quality Assurance Issues:
a. Pipettors calibrated frequently, last calibration date: 11/20/06 Calibrated twice per
year.
b. Major pieces of equipment on service contract:
1. Sorvall RC5B, ABI RealTime PCR, BioRad FPLC
2. Genepix Microarray Reader
b. All genetic constructs confirmed by DNA sequencing both strands, typically with
IDT (Houston TX)
c. PCR QA: rxn controls in each experiment. Always use wildtype strain as a
control so looking for differences in fragment size between mutant and wildtype.
Then pull the mutant insert out of the gel and get it sequenced.
L. Next UTSA Tech Call:
a. March 20, 2007, noon-1pm MT, 1-2pm CT, 2-3pm ET
M. ASM Meeting: Karl will present talk on Ft vaccines at ASM meeting next week.
a. Rick- Karl needs to give funding credit to NIAID. Should Karl describe the
vaccine approaches or not?
b. Vicki: can discuss the scientific advances made but avoid PR.
c. Karl: will present his work as part of vaccine consortium. Karl will describe
UTSA’s role in the overall project and then describe the specific mutations that
UTSA is interested in.
d. Karl: Could Rick give an overview slide? Vicki- yes, okay from Statement of
Work for overview to show where UTSA fits in. Karl won’t describe other
participants’ work in detail. Action: Barbara will send Rick’s old slides to Karl
from the kickoff TVDC meeting.
e. Vicki: A private company currently is saying that it is associated with NIH, on a
tularemia study. Karl would like to avoid giving information to this company.
Company actually sells information that they gather from scientists, who are less
aware of the company’s intent. It is like industrial espionage. Company may be
at the biodefense meeting.
f. Vicki may attend a few sessions, but hadn’t planned
g. Vicki: give credit for support, but since is a verbal presentation, UNM and NIAID
don’t need a summary in advance
h. Action:Karl will share the new slides about a day before the presentation during
the week of 2/26. Karl has a blackberry and we can email him questions even
the day of the meeting.