IGEM:IMPERIAL/2007/Projects/In-Veso/Notes
In-Veso Gene Expression: Notes
Timeline
Week 4
Mon: Order reagents
Tue: -
Wed: Make gene construct of T7 and pLux promoters, Make S30 extract, Make S12 extract
Thur: Make gene construct of T7 and pLux promoters, Make S30 extract, Make S12 extract
Fri: Make gene construct of T7 and pLux promoters, Make S30 extract, Make S12 extract
Week 5
Mon: Test if S30 or S12 is better for the respective strains of E.coli
Tue: Carry out temperature and pH tests on in vitro and in veso extracts
Wed: Carry out temperature and pH tests on in vitro and in veso extracts
Thur: Carry out temperature and pH tests on in vitro and in veso extracts
Fri: Carry out temperature and pH tests on in vitro and in veso extracts
Proposal for Lab
We will first work on making gene constructs and cloning them in E.coli. Restriction digestion, ligation and transformation will be used. Agarose gel electrophoresis will then be used to test for products formed. Cell extracts will be bought from Molecular biology suppliers. Miniprep will be performed to extract plasmids from the cells. The plasmids will then be used to express proteins in vitro and synthethic vesicles, and the proteins tested for by using a fluorometer. Vesicles will be made with phospholipids bought from a supplier. We also need to test these systems for their temperature and pH ranges, as well as their lifespans. All these will use standard equipment in the lab.
