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CollinsMod: A Week of Disaster

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

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CollinsMod: A Week of Disaster

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  1. 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

  2. UV-Induction Assay (I) • Strains: • MC4100 (lacI-) w. pWG-only • MC4100 (lacI-) w. pWG + pTS • Day 1: • Inoculate in 5ml selective LB overnight. • Day 2: • Inoculate 100ul cells in 5ml selective LB w. 2mM IPTG overnight. • Day 3: • 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.

  3. UV-Induction Assay (II) • Day 4: • Backdilute until OD600 ~ 0.3. • Spin down 1ml cells. • Resuspend in 25ul LB. • Examine 1ul on slide. • Microscope • Objective: 100x, oil-immersion • Filters • Bright-field: DIA-DLL • Fluorescence: FITC

  4. Results • pWG-only • 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.

  5. Results: UV-Induction pWG-only: 0 J/m2 pWG+pTS: 0 J/m2 pWG+pTS: 24 J/m2

  6. 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.

  7. 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.

  8. pTS Ptrc l cI lacI PL* 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). • 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.

  9. 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.

  10. 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

  11. 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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