User:University of Washington/30 June 2008

BioBrick Promoter Measurements
- None of the cell cultures plated on Friday showed any growth over the weekend, with the exception of the cells that had been previously transformed and shipped separately. It is possible that the TOP10 cells were no longer competent after the shipping process, or there was an error during preparation.

- Aliquots of TOP10 E. coli were obtained from an Invitrogen kit, in the hopes that the prior difficulties with transformation would be resolved with these new cells. The TOP10 cells were thawed on ice.

- Spotted filter paper sections were removed for the fully-assembled standard reference promoter-RBS-GFP gene-backbone plasmid (I20260), and three different preassembled promoter test constructs (J23150, J23151, J23102).

- Filter paper sections were soaked in 5 uL of TE buffer for twenty minutes, then centrifuged at 14,000 RPM for 3 minutes.

- 50 uL aliquots of the untransformed cells were transferred into five different tubes.

- 2 uL each of plasmid solutions I20260, J23150, J23151, and J23102 were added to four separate aliquots of TOP10 cells. 1 uL of an Invitrogen control plasmid (pUC19) was added to a fifth aliquot.

- The TOP10-pDNA mixtures were incubated on ice for 30 minutes, then incubated in a water bath at 42 degrees Celsius for 30 seconds, then transferred back to ice.

- 250 uL of SOC media (heated to 37 degrees Celsius) was added to each of the TOP10 tubes, then incubated at 37 degrees Celsius on a rotator for 1 hour.

- 133.7 uL of the each of the BioBrick transformed cells were placed on Kanamycin plates, and the control plasmid was placed on a TSY plate free of antibiotic. These plates were incubated at 37 degrees Celsius overnight.

LuxI Cloning (Bryan)
- Electroporation of C0161 LuxI into MG cells on preceding Friday failed. Repeated today, adding a 3 minute microcentrifugation step to improve DNA extraction from filter paper. Used SOC broth instead of TSY. Streaked on plate for tomorrow.

RP4 Cognate Plasmid (Bryan)
- Searched research databases for literature on the genetic regulation of conjugative transfer in the RP4 plasmid. Once key regulatory sequences are identified, we will attempt to modify them so that conjugation can be signalled by exogenous AHL.

LuxR from AraC and TetR
- Run gel for AraC R0080 plasmid 15 ul DNA + 3 ul loading dye, found fragment around 1.5 kb line. (AraC was expected to have 2228 bp)

- List and find what are needed for Quikchange Mutagenesis Reaction. Finalized insertion sequences. Ingrid designed the primers and is ordering them.

LuxR from pLac
- Experiments carried out on J04430 transformed e.coli. Assay of culture showed flourescence of RFP, not GFP (what the part should have contained). Thus, part J04430 is not the right part.

- Overnights of R0010 and J04430 started for miniprep on Tuesday.

Team:University_of_Washington/Notebook