* = On hold. Hypothesis 1: Hypothetical translated region, which is now labeled gene L, confers a deleterious growth phenotype to E. coli. Experiment 1: Compare growth rates of +L and -L strains. Result: Hypothesis 1 confirmed. Exponential growth rates the same on plate reader at ~30 min, but deleterious growth for +L in transition from exponential phase to stationary phase. Quantitatively, saturation for +L is OD600 ~1.6 and occurs at ~9.6hr, whereas saturation for -L is at 2.1 and occurs at ~8.5 hr. Results are reproducible. Followup: Take image of +L and -L E. coli colonies on plate. ---------- * until pBEST-PX-UTR1-deGFP-T500 constuction completed. Characterization A: Characterize PHIX174 promoters with MGapt. Experiment A: Cell free expression of PHIX174 promoters from pBEST-PX-MGapt-T500 at 5 nM (PX = PA, PB, PD, PF, PG, PL, PL-L-PA, promoterless). Follow with expression of each promoter over a range of plasmid concentrations (nM) - 10/2^n, where n = 0, 1 … 6. Constructions: pBEST-PX-MGapt-T500 (set of 8) (completed) .0 pBEST-OR1OR2Pr-UTR1-T500 (completed) Result: --------- * Hypothesis A1: PHIX174 expression regulates one or more PHIX174 promoters. Experiment A1: Cell free expression of PX-MGapt at fixed concentration in presence and absence of PHIX174 ccDNA at 1:1 ratio. Result: ---------- * Hypothesis A2: Observed large relative expression of PL-L-PA-MGapt is due to lower degradation rate of L-MGapt mRNA compared to MGapt mRNA. Experiment A2: Cell free expression of PX-MGapt constructions (specifics fixed by Characterization A result) for 1hr followed by tagetine transcription disruption . Measure degradation rates of mRNA species on the plate reader. Result: ---------- Characterization B: Characterize PHIX174 promoters with UTR1-deGFP. Experiment: Cell free expression of pBEST-PX-UTR1-deGFP-T500 over same concentrations as in CA. Followup with transcription and translation stop experiments and measurement degradation constants. Compare expression rates with CA. Constructions: pBEST-PX-UTR1-deGFP-T500 (first set of clones failed, reconstructing) pBEST-OR1OR2Pr-UTR1-deGFP-T500 (positive control) (completed) Result: ---------- * Characterization C: Characterize PHIX174 promoters with UTRX-deGFP. Experiment: Cell free expression of pBEST-PX-UTRX-deGFP-T500 over same concentrations as in CA. Followup with transcription and translation stop experiments and measurement degradation constants. Compare with result of CB to characterize the effect of the native PHIX174 UTRs on expression. Constructions: pBEST-PX-UTRX-deGFP-T500 (set of 8 constructions as in CA and CB) pBEST-OR1OR2Pr-UTR1-deGFP-T500 (positive control) Result: ---------- Hypothesis 2: Gene L is necessary for phage propagation. Experiment 2: Use cell-free expression to produce phage from WT ccDNA and geneL-L21STOP ccDNA. Count number of active phage infections on cell plaques in triplicate. Constructions: Using PFU Ultra DNAP, construct the following by whole plasmid PCR. 1. PHIX174 WT ccDNA 2. PHIX174 T3845A ccDNA (geneL L21STOP) Notes: Several attempts at PCR have failed. Primers seem to self-hybridize and elongation along the plasmid template is not occuring. Latest attempt is using the method described in Chen and Ruffner 1998. Result: ---------- Hypothesis 3: Gene L codes for a ~6 kDA protein. Experiment 3: Simga28 cascade cell free expression in vesicles in batch mode of +L and -L followed up by high density SDS page. Constructions: pBEST-OR1OR2Pr-UTR1-simga28-T500 (amplified) pBEST-Ptar-UTR1-deGFP-T500 (amplified) pBEST-Ptar-UTR1-eGFP (in stock) pBEST-Ptar-UTR1-L-T500 (amplified) pBEST-Ptar-UTR1-L-eGFP-T500 (amplified) Result: Preliminary results show binding of L-eGFP fusion to artificial vesicle membrane. ---------- * Hypothesis 4: Gene L complexes with membrane. Experiment 4: Cell free expression in vesicles of H3 constructions. Followup with in vivo expression in cells observed by microscope (use PT7 promoter). Constructions: Use PIvex2.3d (PT7 and T7term included) Result: ---------- * Hypothesis 5: Level of gene L expression modulates deleterious effects of protein L. Experiment 5: Using IPTG inducible pT7-L, pT7-deGFP, pT7-L-deGFP fusions, observe cell free expression and in vivo growth rate / saturation point over variable induction levels. Investigate whether deleterious effects scale with level of gene L expression. Constructions: similar to H4.