Engineering Enzymes Flashcards

1
Q

Site Directed Mutagenesis

A

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

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2
Q

Subtilisin SDM

A

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

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3
Q

DNA Shuffling

A

mix, digest, melt, anneal, extend

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4
Q

Screening

A

test everything

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

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5
Q

Directed Evolution of Lipase

A

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

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6
Q

Saturation Mutagenesis

A

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

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7
Q

CASTing

A
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
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