Lactose Summary
We want to make two plasmids:
Plasmid #1 - A constitutive promoter with LacZ and LacY.
Plasmid #2 - A lactose inducible promoter with lysozyme and holin.
The general idea is to overexpress LacY (Lactose transporter) with LacZ (Beta-Galactosidase). When lactose is present, the cell will uptake lactose and will flip on the Lac promoter repressed by lacI when enough lactose is present. We then express lysozyme and holin which will lyse the cell releasing beta-gal cleaving lactose into glucose and galactose. Hopefully, glucose and galactose will be taken up by the host in the colon.
Lactose Outline
6.19.08
We have decided to use the I732017 LacZ (1009/11H) because we could not transform I732005 LacZ (1018/6F).
To double check, we will plate our transformed I732005 LacZ with 100μL onto a kan plate, and the rest on an Amp plate.
We ordered primers for LacY, and we will need to PCR out LacY. We then need to run site directed mutagenesis on LacY by added 2H to prevent EIIA(GLUC) binding.
For our constitutive promoter, we will use various strengths to test our beta-gal concentrations.
Low expression - J23113
Medium expression - J23106
High expression - J23100
We need to cut out mRFP, which is originally in the promoter plasmid family of J23100, by doing a digest with SpeI and PstI. We will paste in LacZ and LacY, and add a double TT.
6.20.08
Colonies grew from I732017 LacZ transformation. They will be cultured Sunday afternoon.
LacY, LacY +H, and LacY +2H has been inputted into the registry of parts. Primers for LacY site directed mutagenesis were designed.
When primers for LacY arrive, need to PCR LacY out of E. coli genomic DNA.
After LacZ culture is grown, mini prep DNA.
6.22.08
Set up o/n cultures.
1. LacZ in Amp
2. pSB2K3 in Km
3. p1005 in Amp
4. p1005 in Amp + Tet (Just to test the strain is both Tet and Amp resistance)
6.23.08
Mini prep'd LacZ and pSB2K3, eluted in 50 μL.
LacZ conc. = 115.4 ng/μL
pSB2K3 conc. = 427.4 ng/μL
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