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 cite 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.
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