Biochemistry 2280A Lecture Notes - Lecture 22: Dna Ligase, Gene Knockout, Dna Profiling

• School: Western University
• Department: Biochemistry
• Course: Biochemistry 2280A
• Professor: Chris Brandl

Document Summary

Expressing yfp in e. coli: coli are the most commonly used organism for expressing yfp (protein) Introns are found in human genes: human and e. coli promoter structures are different, different mechanisms of translational initiation, different post-translational modifications occur in prokaryotic and eukaryotic cells, ex. If in eukaryotes there is phosphorylation, then it might not be expressed properly in eukaryotes. Expressing yfp in e. coli: use expression plasmid, coli promoter sequence, human gene is downstream from e. coli promoter and ribosome binding site (e. coli, choice of plasmid. Producing yfp: purify protein from e. coli, check protein for purity and activity, ready to use. Site directed mutagenesis: creating a gene with altered sequence and in turn, altered function. Custom designing yfg: converting a codon for asp to a codon for ala, gac to gcc= single base change, separate strands to obtain single stranded dna (open it up) Synthetic oligonucleotide primer containing desired mutated sequence (gcc)