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Sensitivity analysis of gene knockout mutant library was further performed (phenomics data).

Thus, high-throughput screening of PHB synthase mutant library was available using LipidGreen1 through 96-well microtiter plate-based assay.

Previously cloned BGL1 encoding β-glucosidase I (BGLI) was subjected to error prone PCR and the mutant library was cloned in pPIC9 followed by transformation into Pichia pastoris.

Another "Super Pgap" mutant library was further constructed where the strong native promoter Pgap of Z. mobilis was randomly integrated throughout the genome of Z. mobilis 33C via an in vitro transposon mutagenesis system.

Three mutants (SA8, SA17 and SA20) were isolated on indicator plates (i.e., 1% phosphatidylcholine gels containing 30% dimethyl sulfoxide (DMSO)) after a second mutant library was treated in 50% DMSO for 36 h.

This protocol comprised the following: (i) a linear DNA mutant library was generated by error-prone PCR or shuffling, and a linear vector backbone was prepared by regular PCR; (ii) the DNA multimers were generated based on these two DNA templates by POE PCR; and (iii) the one restriction enzyme-digested DNA multimers were ligated to circular plasmids, followed by transformation to E. coli.

The methods that are involved in creating a mutant library are extensive and can be divided into several categories according to their basic ideas.

A novel and effective mixture culture method was proposed and used to realize the whole mutant library being high-throughput screened after mutagenesis.

Genetic diversity creation is a core technology in directed evolution where a high quality mutant library is crucial to its success.

Two of the transposon mutants, ΔrsmB (Ech148) and ΔgacS (Ech149), identified in the mutant library were used in this study.

TG1 cells containing phagemid coding a scFv, C-CPE, C-CPE mutant or C-CPE mutant library were culture in 2YT medium containing 2% glucose and ampicillin.