Transcript GroupMeeting(BioSketch)-CollinsMod-_2005-07

BioSketch
Harvard iGEM 2005
CollinsMod: A Week of Disaster
Testing the Collins Circuit
MC4100 (lacI-) cells co-transformed w. pWG + pTS
Unable to reproduce results of Kobayashi paper w.
respect to UV induction
Building constructs
Mutations in the parental pTS plasmid
Failures w. pEL (lacI-only construct) and derivatives
Cloning of reporter constructs
PCRs successful
Subsequent steps unsuccessful
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Harvard iGEM 2005
Strains:
UV-Induction Assay (I)
MC4100 (lacI-) w. pWG-only
MC4100 (lacI-) w. pWG + pTS
Day 1:
1.
Inoculate in 5ml selective LB overnight.
Day 2:
1.
Inoculate 100ul cells in 5ml selective LB w. 2mM IPTG overnight.
Day 3:
1.
2.
3.
4.
5.
Backdilute 100ul cells in 5ml fresh selective LB and grow until
OD600 ~ 0.3.
Pipet 25ul cells onto 3cm agar plates.
Incubate @ 30C for 2h.
Irradiate @ 0, 12, 24J/m2 UV in crosslinker.
Scrape cells into 5ml fresh selective LB and grow overnight.
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Harvard iGEM 2005
Day 4:
UV-Induction Assay (II)
Backdilute until OD600 ~ 0.3.
2. Spin down 1ml cells.
3. Resuspend in 25ul LB.
4. Examine 1ul on slide.
1.
Microscope
Objective: 100x, oil-immersion
Filters
Bright-field: DIA-DLL
Fluorescence: FITC
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Harvard iGEM 2005
pWG-only
Results
Clearly fluorescent.
Almost every cell.
pWG+pTS
No fluorescence.
Cells somewhat dumpy compared to pWG-only cells.
IPTG Treatment
Made no difference.
UV Irradiation
Made no difference.
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Results: UV-Induction
pWG-only: 0 J/m2
pWG+pTS: 0 J/m2
pWG+pTS: 24 J/m2
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Regulation by lambda CI
pWG+pWCI transformants (without IPTG or UV)
give no fluorescence.
Digests of those transformants clearly indicates
that pWG is present.
pWG-only controls glow.
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Harvard iGEM 2005
Discussion of Microscope Experiments
We have not been able to replicate the results of
the Collins lab.
We did not use the same strain, however, and it is
possible that there is an endogenous factor that is
repressing the expression of the GFP … although
only when the toggle-switch vector is present.
We are deleting the lacI and lambda-cI regions
from the pTS vector to get just the backbone to
see if the same result (no fluorescence) is found
when co-transformed w. pWG.
We have not heard from Collins regarding the
strain request.
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Harvard iGEM 2005
Mutations in the parental pTS plasmid
Every sequence of the original pTS or constructs derived
from pTS (e.g. pWCI, pTS241, pTS265) have four
mutations:
Two substitutions in the cI-regulated promoter of lacI
One substitution in the lacI-regulated promoter of cI
One frame-shift deletion in the cI coding region (where there
should be a stretch of 6 As, there are only 5).
lacI
PL*
Ptrc
l cI
pTS
The mutations are surprisingly close to each other
All four within 200bp.
The two in lacI promoter are three nucleotides apart.
Substitution mut in cI promoter and the deletion in coding
region are less than 20 nucleotides apart.
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Implications of the mutations
The most problematic is the deletion, which, if
true, introduces a stop codon 7 amino acids after
the start.
Nonfunctional cI wouldn't explain the results of
the UV-induction experiments, however.
Getting the pTS backbone (pTV) is thus crucial.
If the deletion is really there, then
pTS
pTS241
pTS265
pWCI
must be mutagenized to add a nucleotide.
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Harvard iGEM 2005
Cloning Experiments
lacI-only constructs (pELc; pEL241; pEL265)
Ligations gave transformants, but analytical digests did not give
expected bands
Previously: Klenow rxn, then CIP; 15 or 30min ligation
Now: CIP, then Klenow; 1h ligation
mCherry reporter for cI regulation (pWCh)
PCR of mCherry from the plasmid given by MIT successful
Analytical digests of transformants suggest
Incomplete digest
Recircularization
CIP failure
GFP reporter for lacI regulation (pEG)
PCR of lacI-regulated promoter P(trc) successful
XmaI/EcoRI digest of PCR product and pWG vector problematic
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This week with CollinsMod
Confer w. Collins regarding
The mutations
The failure to replicate results
Consider alternative cloning strategies for pELc
and derivatives
Site-directed mutagenesis to remove 2nd BamHI site?
Get real dam- cells?
Retry ligation for pWCh
Retry digestion for pEG
Use of LacZ for assaying regulation?
Like a ratchet.
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