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After three rounds of mutagenesis, a mutant was generated that decreased the biocatalyst loading 3-fold.
09N1-I149V mutant was generated by using primer F: 5′-CATTCCAATGGAACCGTTAAAGACAGGAGC-3′ and primer R: 5′-GCTCCTGTCTTTAACGGTTCCATTGGAATG-3′.
The hyperactive mutant was generated by introducing two point-mutations into the cytosolic flexible interface between the two RCK domains of the wild-type BKCa channel.
viciae AapQ site directed mutant was generated as follows.
In parallel, a constitutively dimeric mutant was generated (R227E/K228E) [36].
The hda1Δ/Δ mutant was generated by sequential gene deletion using auxotrophic markers (Table 1).
The CF2[KG08941-R3] mutant was generated by P-element excision of the CF2[KG08941] stock.
An isogenic rho0 mutant was generated by ethidium bromide treatment as described previously [39].
The C-terminal deletion mutant was generated by deleting the 27 aa at the C-terminus.
The ΔhemO::gm mutant was generated by amplifying the altered hemO fragment from IA614 [35].
The cofilin-1 mutant was generated via a proviral insertion into the cofilin-1 gene.
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