Engineering Enzymes

Question 1

Site Directed Mutagenesis

Answer 1

PCR mediated
design oligonucleotide primers with codon change in the middle
gene into plasmid
methylated non-mutant DNA digested

Question 2

Subtilisin SDM

Answer 2

mutate glycine to larger amino acid
chemically modify mutants
introduce hydrophobicity (sulfur)

Question 3

DNA Shuffling

Answer 3

mix, digest, melt, anneal, extend

Question 4

Screening

Answer 4

test everything

facilitated: what you want to test looks different
selection: what you want to test is the only thing that survives

Question 5

Directed Evolution of Lipase

Answer 5

resolve enantiomers
want high e value
change one amino acid at a time
sequential mutants increase e

Question 6

Saturation Mutagenesis

Answer 6

determine structure
choose 4 amino acids in active site
mutate to every other codon
best mutants in 1st/2nd shell

Question 7

CASTing

Answer 7

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