User:University of Washington/7 August 2008

Yeast Shuttle Plasmid
- Obtained yeast strain K. lactis

- Obtained primers for amplification of LAC4-LAC12 region of K. lactis genome

- PCR'd the K. lactis with primers

- Made glyercol stocks of BBa_J63005 and BBa_J33202

BioBrick Promoter Measurements
- Inoculated overnight cultures with colony isolates of TOP10 for promoter constructs I20260, I20259, I20261, I20267, and empty TOP10 cells.

LuxR from AraC and TetR(Faifan)
- Both ligated DNA(Elowitz's promoter + pSB1AC3) transformation grew on Amp. Got single colonies. Picked and overnight.

- Continued QuikChange
 * Added 1 ul dpn1 in reaction 1, 2 and negative control, mixed and span 1 min.
 * Incubated 37 degree Celcius for 3 hours
 * PCR purified
 * Transformed 2 ul of each into XL1-Blue, grew on Amp plate

MG1655Z1(Faifan)
-Diluted 1:100 of overnight culture of MG1655Z1(2 colonies from Tsy#2 plate) in Tsy broil.

-Incubated the dilution 3 hours

-Diluted 1:100 again in Tsy, inoculated in Tsy plates.

Lambda Red Recombineering of RP4 (Bryan)
Attempted co-electroporation and recombineering of RP4 plasmid with Cm cassette in DY331. Performed three trials with 10 ng, 20 ng, and 25 ng of RP4 to determine optimal quantity of target plasmid for recombination. Also included control transformation without recombinant cassette. There was growth in the control transformation, but the recombinant transformations failed. Since optimizing the quantity of target DNA was not helpful, subsequent experiments will attempt to optimize the quantity of insert DNA.

Made glycerol stock of IS010, in case we need to amplify the tet resistance cassette in the future.

Conjugation (Scott)
Conjugation (Bac-Yeast) protocol #2 - pAC88 neg control - pAC88 w/ pUB307

All bacteria began conjugation at OD600 of .15(~3E7/mL) All yeast began conjugation at OD660 of .1(~1E7/mL)

1. 250uL bac w/ 250uL yeast 2. 200uL w/ 300uL 3. 100uL w/ 400uL 4. 50uL w/ 450uL

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