LIFESCI 3 Lecture 28: Lecture 28 cloning II
Document Summary
Lecture 28 insulin: e. g. ampicillin + unregulated lac z or dual antibiotics, grow selected bacteria on agar/liquid culture, affinity chromatography. Isolate some plasmid dna & digest (length of plasmid vs insert) Reporter assay: take regulatory region of gene -> put in plasmid, but ahead of promoter (not directly downstream of promoter) to test how the sequence regulates promoter. Unnecessary to have natural gene regulated by regulatory sequence downstream of promoter. Put something visual: reporter vector; e. g. gfp or luciferase (fluoresce proportional to the amount of gene expression driven by regulatory sequence) Luciferase has lots of restriction enzyme sites -> helps clone in a regulatory region upstream of a promoter. E. g. promoter bashing: mutate a promoter sequence & test how mutation influences promoter function. Crispr: crispr = enzyme primitive immune system in bacteria. Can express it in any cell type: cas9 = endonuclease; can cut anywhere in dna. Driven by sgrna; contains complementary sequence to what cas9 cuts.
