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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 1 BioSketch 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. 2 BioSketch 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 3 BioSketch 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. 4 BioSketch Harvard iGEM 2005 Results: UV-Induction pWG-only: 0 J/m2 pWG+pTS: 0 J/m2 pWG+pTS: 24 J/m2 5 BioSketch Harvard iGEM 2005 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. 6 BioSketch 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. 7 BioSketch 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. 8 BioSketch Harvard iGEM 2005 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. 9 BioSketch 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 10 BioSketch Harvard iGEM 2005 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. 11