By Frances Hamilton Arnold (Editor), George Georgiou (Editor)
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Additional resources for Directed Enzyme Evolution: Screening and Selection Methods (Methods in Molecular Biology Vol 230)
The total mRNA is extracted, and the gene for T7 RNA polymerase is reverse-transcribed and PCR-amplified. The gene fragments containing sequence variations (shown as *) are re-cloned and re-transformed. Several rounds of selection and amplification lead to the accumulation of polymerase variants with altered promoter specificities. ). The autogene library is initially plated on LB agar plates without IPTG. Colonies are lifted via nitrocellulose filters to a new plate containing IPTG and protein expression is induced.
Binding of the lac repressor, to the lac operator effectively blocks transcription by T7 RNA polymerase. Addition of IPTG derepresses the T7 RNA polymerase promoter and induces the expression of the T7 RNA polymerase gene. The plasmid pET28a also contains the T7 terminator and a ribosomal binding site for translation of the cloned gene. The steps in the construction of the wild-type T7 autogene are listed below: 1. 1. 1: GGG AAT TCT TAC GCG AAC GCG AAG TCC GA (EcoRI site is underlined) 2. 1 (Topo TA cloning kit, Life Technologies) using the protocol suggested in the kit (see Note 2).
At the conclusion of the self-amplification process, the variants could be thrown together, and polymerase mRNAs should be roughly represented in the mixed population according to the enzymatic success of the polymerases they encoded. In the case of a library cloned behind a mutant promoter, mRNA extracted from the population of cells should represent polymerase variants in rough proportion to their ability to utilize the mutant promoter. Re-cloning the successful sequences should over-represent successful polymerases relative to unsuccessful polymerases, and provide a means to carry out iterative rounds of selection and amplification.