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User:University of Washington/23 July 2008
From 2008.igem.org
LuxR from AraC and TetR
- Miniprepped AraC R0080
- Results from Transformation of mutated AraC
- Positive control : a lot of growth
- Negative control : some growth (expected NO growth because primers weren't added.)
- Reaction one : some growth
- Reaction two : some growth
- Picked 4 colonies from each reaction and prepared overnight culture.
- Primers for Elowitz's promoter were designed and ordered.
Lambda Red Recombineering of RP4 (Bryan)
Troubleshooting of PCR continued. Attempted PCR on control template at 1.5 mM, 2 mM, 2.5 mM, and 3 mM magnesium chloride. No amplicon at 1.5 mM, which conflicts with results from yesterday's PCR. Best amplicon appeared at 2 mM. Since the results are inconsistent, it is difficult to optimize the reaction concentration based on this experiment.
Mini-prep of pSIM5 had no DNA in eluate. Putative error: pSIM5 does not have cam cassette leading to loss of plasmid or failure to grow in ON culture. Will not repeat mini-prep since pSIM5/host is in glycerol stock and not necessary for immediate plans.
BioBrick Promoter Measurements
- TOP10 cells were transformed with 2007 parts for BioBricks J63005 and J33202 (a constitutive yeast ADH1 promoter and a bacterial RBS+LacZ part), and 2008 parts for promoter constructs I20261 and I20267.
- The TOP10 cells transformed with J63005 and J33202 were added to an agarose plate with ampicilin, and those cells transformed with I20262 and I20267 were added to a plate with kanamycin.
- A sample from a glycerol stock for a death-resistant DB3.1 strain with the P1010-pSB3K3 plasmid was plated on a kanamycin plate, and a strain of DH5 alpha with a LacI mutation were added to a plate consisting of LB media wit no antibiotic.
- Glycerol stocks for TOP10 cells with promoter construction I20260, I20268, I20269, and I20270 were added to kanamycin plates.
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