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Expression Plasmids and Hosts

The Usual System. E. coli hosts genetically modified to be capable of producing recombinant proteins.Genes/segments, cloned into plasmids containing bacteriophage T7 transcription (sometimes translation) signals. These are the source of the T7 RNA polymerase provided to the host cell to start exp

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Expression Plasmids and Hosts

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    1. Expression Plasmids and Hosts The down and dirty daily dealings of the NCSB Lab

    2. The Usual System E. coli hosts genetically modified to be capable of producing recombinant proteins. Genes/segments, cloned into plasmids containing bacteriophage T7 transcription (sometimes translation) signals. These are the source of the T7 RNA polymerase provided to the host cell to start expression of the target protein, and of the target gene for expression. Transform the plasmid into the host and there you are…………………..if you’re lucky.

    3. Protocol for Transforming E. coli with a plasmid Thaw an aliquot of CaCl2 competent cells on ice for up to 15 minutes. (Or use freshly made competent cells.) Add 10-50 ng of plasmid DNA vector containing the gene of interest to a 50 µl aliquot of the cells. Mix by flipping, gently. Incubate the tube on ice for about 20 minutes. Place the tube at 42°C for 2 minutes, then put it back on ice for 3 minutes. Add 400 µl of SOC medium and incubate the tube for between 15-30 minutes at 37°C. Plate the transformed cells on two LB-agar plates, one at incubation concentration, one at 10X. Incubate the Petri plates at 37°C for 20±4 hours.

    4. The Happy Result

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