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Challenges in Replicating UV-Induction Results with Collins Circuit Constructs

This report details the ongoing difficulties encountered in reproducing the results from the Kobayashi paper regarding UV induction using the Collins Circuit in MC4100 (lacI-) cells. Key experiments involving the co-transformation of plasmids pWG and pTS failed to yield fluorescence, raising concerns about possible mutations in the parental pTS plasmid and its impact on gene regulation. Techniques employed included PCRs, cloning of reporter constructs, and successive UV irradiation assays. Discussion emphasizes the importance of addressing plasmid mutations and exploring alternative cloning strategies.

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Challenges in Replicating UV-Induction Results with Collins Circuit Constructs

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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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