Cloning
into pASK
vector
Transformation
to E. coli
3
CH
M.HaeIII
mutated ORF
InDels and random
point mutations
library
High
throughput
sequencing
CH3
Amplify by
error-prone
PCR
Extracted naive
plasmids pool
CH3
CH3
High
throughput
sequencing
Map the occurrence
of mutations
irrespective of
selection
G0 - the naïve
repertoire
CH3
CH3
X 17
rounds
Extracted
selected
plasmids pool
Map the repertoire
of tolerated
mutations
G17 - the selected
repertoire
Selection by
HaeIII
digestion
3
CH
CH3
Digested fragments of
non-active MTases
(non-tolerated mutations)
CH3
Retransformation
Intact plasmids
expressing active
MTases
(tolerated mutations)
CH3