Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening.

ABSTRACT: The Golden Gate strategy entails the use of type IIS restriction enzymes, which cut outside of their recognition sequence. It enables unrestricted design of unique DNA fragments that can be readily and seamlessly recombined. Successfully employed in other synthetic biology applications, we demonstrate its advantageous use to engineer a biocatalyst. Hot-spots for mutations were individuated in three distinct regions of Candida antarctica lipase A (Cal-A), the biocatalyst chosen as a target to demonstrate the versatility of this recombination method. The three corresponding gene segments were subjected to the most appropriate method of mutagenesis (targeted or random). Their straightforward reassembly allowed combining products of different mutagenesis methods in a single round for rapid pro

SUBMITTER: Quaglia D 

PROVIDER: S-EPMC5298319 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature