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

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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
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.