IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/08/13

UNAM-Genomics-Mexico team

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Working on E.coli trpR mutant I repeated the PCR colony. Using the same previously described protocol. Results:E.coli trpR mutant Samples: The trpR mutant colonies that were selected to make the colony PCR were: 2,3,9. Colony control: E.coli k12 wt. PCR colony trpR-. Lane1:Ladder. Lane6:E.coli k12 wt. Lane7:trpR- colony 3. Lane8: trpR- colony 9.The other lanes are samples from other experiments. The sizes of the PCR products are around the expected lenght - 421 nt - for E.coli k12 wt and trpR mutant colony 9. So, these PCR products will be used to sequence and analyse the trpR frameshifth mutation. Working on LovTAP promoters: Restrictions Strong Promoter J23102 Once the cells were correctly transformed with the plasmid J61002 harboring the strong promoter J23102, I have started the plasmid extraction procedure. Plasmid J61002 with promoter J23102: The plot shows the enzyme restriction cutting sites inside Plasmid J61002 and the location of the following elements: promoter J23102, RFP protein, AmpR-ampicillin resistance- and the origin of replication. Plasmid extraction Protocol I am using the High Pure Plasmid Isolation kit from Roche Restriction enzyme assay:Preparation for ligation After finishing the isolation of plasmid J61002, I have started the restriction enzyme assay in order to remove the RBS site, the RFP gene and the doble terminator from plasmid J61002 to replace them with LovTAP gene. Restriction enzymes Mixture: Restriction enzymes: SpeI and PstI. Reactive Quantity Plasmid DNA 15μL Buffer 2 4μL BSA 1μL Enzymes 2μL for each one. HPLC Up to a final volume of 40 μL of the mixture Results:LovTAP Promoters-Restriction enzyme assay-Preparation for ligation The restriction reactions were incubated at 37°C overnight. Note: Inactivate the enzymes at 80°C during 10 min. According to the next image, the two bands observed in Lane 4,5 and 6 are around the expected sizes that should be 887bp and 2096bp, considering the cutting pattern using SpeI and PstI enzymes. Restriction reactions. Lane1:Ladder. Lane4: Plasmid J61002 colony 4. Lane5:Plasmid J61002 colony 5. Lane6:Plasmid J61002 colony 6. The other lanes are samples from other experiments. Working on LovTAP synthesized plasmid from Mr. Gene Mariana successfully transformed the LovTAP synthesized plasmid, so that I am going to start the plasmid extraction procedure. Plasmid extraction Protocol I am using the High Pure Plasmid Isolation kit from Roche Results:LovTAP synthesized plasmid from Mr. Gene Mistake: I grew up the cells using a wrong antibiotic (ampicillin), so they didn't grow up. I will repeat the experiment using kanamycin antibiotic.