UNM TVDC: UTSA Tech Call Minutes 3/20/2007
Prepared 3/23/2007: Barbara Griffith
Sent to UTSA for review: 3/23/07
Reviewed: Karl Klose (ADD DATES) and Bernard Arulanandam (3/23/07)
Distributed to NIAID on: (ADD DATE)
Present: Karl Klose, Barbara Griffith, Vicki Pierson, Marlene Hammer, Bernard Arulanandam,
Rick Lyons
Absent: Joe Breen, Kristin DeBord
Status of January action items:
 MS 16, 39 and 48 Completion Reports: All 3 have been received by UNM and need
revisions. On 2/1/07, Barbara requested edits on MS#16 Completion report. On 3/21/07, Karl
agreed that Barbara could work directly with Jeff Barker to complete the MS#16 Completion
Report
The meeting was recorded for the purposes of the minutes.
A. SLIDE 2: 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. SLIDE 3: Milestone #43 Flow Chart: Creation of uvrA and uvrB mutant F. tularensis
subsp. holarctica (LVS) strains
a. Have constructed the plasmids which will be used to mate into LVS
C. SLIDE 4:Milestone #43: Creation of uvrA and uvrB mutant F. tularensis subsp.
holarctica (LVS) strains
a. Ping Chu replaced Jirong Liu (Note: Barbara received Dr. Chu’s C.V. on 3/23/07)
b. Got growth of LVS some NaCl and with 10% sucrose in the media, though 5%
sucrose is in the literature.
c. Have isolated the LVS colonies that are kan resistant and chloramphenicol
sensitive; The Kan gene is in middle of uvr B gene so UTSA has removed uvrB
gene and put the Kan gene in it’s place; need to confirm by PCR and sequencing
that the plasmid backbone has been removed by the counterselection with the
sucrose.
D. SLIDE 5:Milestone 49 Flow Chart: Creation of mutant F. tularensis subsp.
tularensis strains (SCHUS4)
E. SLIDE 6&7 :Milestone #49A: Construction of iglC F. tularensis subsp. tularensis
strain
a. High copy vector with Sac B counterselection approach is similar to literature
and currently screening SCHU S4 clones for the correct plasmid.
b. To test high copy vector technique, are using mglA gene, which regulates an acid
phosphatase which gives the colonies the blue vs white phenotype; mglA gene is
also single copy in the SCHU S4 chromosome. UTSA got the vector into SCHU
S4 no problem; got plasmid backbone to come out of the SCHU S4
chromosome- no problem, which is a step forward because previously without
the sucrose counterselection, the plasmid would not come out. Currently are
screening for colonies off the sucrose plate to identify those retaining mutant
mglA gene in the chromosome.
c. Low copy vector approach: mating vector may be working but no successful
iglC construct yet for sure.
d. Targetron approach: has achieved knockout of beta-lactamase (bla2) gene and
mglA gene in SCHU S4; UTSA knows the targetron system works in SCHU S4.
Have to get protein and RNA based intron expressed in the cell to target to iglC
gene and knock it out. Currently are looking for the targetron insert into the iglC
gene to knock out the iglC gene. The longer the targetron plasmid is in the target
gene, the more times the targetron plasmid will target the iglC gene and will
hopefully knock out both iglC gene copies. Steve is doing the work in Karl’s lab.
e. Rick: Targetron approach has the advantage of knocking out multiple gene
copies.
f. Karl: Targetron requires designing the oligos for the specific gene of interest an
and clone them into a plasmid, but this type of process is required for other gene
knockout approaches anyway. Just put the targetron plasmid in and need a
screening method for the clones containing the successfully targeted gene.
UTSA is growing the targeted SCHU S4 at 30oC rather than 37oC, even though
the bacteria grows faster at 37oC. UTSA adapted the plasmid to have a
temperature sensitive mutation so that the plasmid can be removed afterwards.
Then started growing the bacteria at 30oC rather than 37oC and the efficiency
increased dramatically. Previously, got 3 insertions/100 with 37oC and then got 8
insertions /10 colonies with 30oC. 30oC dramatically promotes intron targeting
system. UTSA hopes that the iglC insertion efficiency is similar to the higher
efficiency found with the bla2 and mglA genes. UTSA deduces that since 8/10
have 1 insertion that a second round will result in 8/10 having a second insertion
too. Steve found a publication that supported the lower temperature dramatically
enhancing the intron targeting system. Plans to give more report next month
F. SLIDE 8:Milestone 51 Flow Chart: Creation of uvrB + pdpD, iglA, iglB, iglC, and iglD
mutant F. tularensis subsp. novicida double mutant strains
G. SLIDE 9:Milestone #51: Creation of uvrB + pdpD, iglA, iglB, iglC, iglD mutant F.
tularensis subsp. novicida strains
a. Have made the iglD/uvrB double mutant in F novicida
H. SLIDE 10:Milestone 50 Flow Chart: Immunologic characterization of F. tularensis
subsp. novicida, subsp. tularensis, and LVS strains
a. iglB mutant of F novicida is highly attenuated in mice- shown in last month’s
report
I.
SLIDE 11: Milestone #50A: Immunologic characterization of F. tularensis subsp.
novicida, subsp. tularensis, and LVS strains- results update
a. Evaluating the protective efficacy of the F. novicida iglB mutant as a vaccine
candidate in Balb/C mice.
J.
SLIDE 12: Milestone 50A- figure 1, protective efficacy of iglB immunization against
F novicida infection.
a. Were immunized with F novicida iglB mutant, rested for 30 days and then
challenged with 100x LD50 F novicida
b. All vaccinated mice at all doses were highly protected from high dose challenge
of F novicida.
c.
Unvaccinated mice died of the F novicida challenge.
K. SLIDE 13: Immunologic characterization of F tularensis subsp novicida, subsp
tularensis, and LVS strains
a. Analyzing antibody profiles of mice immunized with the Ft novicida iglB mutant,
after vaccination.
b. Coating ELISA plates with uv inactivated iglB sera
L. SLIDE 14: Milestone 50A- Immunologic characterization of F tularensis subsp
novicida, subsp tularensis, and LVS strains – bar graph data slide
a. At all the vaccination doses, detected nice day 14 and day 28 antibody
responses. IgG2A has gone up. So the animals are protected by the Fnovicida
iglB mutant vaccination and the animals make a robust antibody response after
vaccination.
b. Rick: what is the antigen on the plate?
c. Bernard: uv inactivated organisms are used as the antigen on the plates.
d. Bernard: Use a non-specific, unrelated antigen negative control. Hen egg
lysozyme is the non-specific antigen used.
M. SLIDES 15&16: Plan for following month:
a. Milestone #16: completed.
b. Milestone #39: completed.
c. Milestone #48: completed.
d. Milestone #43: have right phenotype and are confirming genotype for uvrB kan
mutation
e. Milestone #49A: in progress
f. Milestone #51:in novicida, constructing uvrB /iglA double mutants
g. Milestone #50: will look at interferon gamma and IL 4 response. Will do a T cell
recall. Will perform lung histology at 30 days after the lethal challenge, to see if
no damage to lungs after protected from F novicida by Fn iglB immunization
N. Additional Discussions not associated with slides
a. Site Visit: Barbara will call Karl at 11MT to discuss the site visit agenda
(completed 3/21/07)
b. Vicki: nice succinct , well organized and easy to follow presentation.
c. Targetron and 30oC - Vicki: since 30oC works for intron targeting and for what
other species are you using 30oC? Vicki: has this 30oC helped for other
approaches?
d. Karl: 30oC works in SCHU S4 and in novicida. Intron targeting system is
destabilized in general in other bacteria like E. coli at 37oC and 30oC stabilized
the intron targeting system. This increased efficiency of insertions with 30oC
seems general and not specific to Francisella. Came into literature just 2 months
ago.
e. Vicki: if you have problems with recombination in other species has the 30 oC
helped?
f. Karl: the 30oC helps specifically with this intron targeting system and not with
recombination in general. The intron targeting system is a protein and an RNA,
like a ribozyme, as the RNA actually folds into a enzyme. The protein facilitates
it to cut the DNA and insert the transcript in. The protein called LTRA is the
temperature sensitive enzyme. Doesn’t know the organism origin of LTRA
enzyme, it could even be from E.coli.
g. ASM meeting attended by Karl: Didn’t see Karl’s slides due to computer being
stolen. Only tularemia people were Tina, Justin Karl. No other people attended
who worked on tularemia. Karl gave a talk and room packed and no questions.
Strange meeting. Chatted with Mark Bolanowski. Used 2 of Rick;s slides for the
overview of the vaccine contract and who was involved and then talked about
iglC vaccination.
O. Schedule: Tech Calls , Site Visit, and Semi-Annual Report
a. Site Visit: 4/9/2007 Monday
b. No Tech calls in April
c. Semi Annual Report is due on April 7, 2007 for the 10/1/06 to 3/31/07 period
d. Next Tech call is May 15, 2007, noon-1pm MT, 1-2pm CT, 2-3pm ET