Engineering Enzymes Flashcards
Site Directed Mutagenesis
PCR mediated
design oligonucleotide primers with codon change in the middle
gene into plasmid
methylated non-mutant DNA digested
Subtilisin SDM
mutate glycine to larger amino acid
chemically modify mutants
introduce hydrophobicity (sulfur)
DNA Shuffling
mix, digest, melt, anneal, extend
Screening
test everything
facilitated: what you want to test looks different
selection: what you want to test is the only thing that survives
Directed Evolution of Lipase
resolve enantiomers
want high e value
change one amino acid at a time
sequential mutants increase e
Saturation Mutagenesis
determine structure
choose 4 amino acids in active site
mutate to every other codon
best mutants in 1st/2nd shell
CASTing
determine structure group amino acids in active site PCR design primer - substitute every amino acid to every other amino acid generate sub-libraries for each region