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Figure 1 | BMC Biotechnology

Figure 1

From: A combined in vitro / in vivo selection for polymerases with novel promoter specificities

Figure 1

Autogene selection. (A) Scheme. An autogene library containing the polymerase pool and promoter mutations is transformed into cells and induced. Active autogenes overproduce T7 RNA polymerase and mRNAs encoding the polymerase. Total mRNA is extracted, and the gene for T7 RNA polymerase is selectively reverse-transcribed and PCR-amplified. The gene fragments containing sequence variations (shown as *) are re-cloned and re-transformed. Multiple rounds of selection and amplification lead to the accumulation of polymerase variants with altered promoter specificities. (B) Screen for active variants. The autogene library is initially plated on LB agar plates without induction. Colonies are lifted via nitrocellulose filters to a new plate with IPTG and protein production is induced. Colonies that have active autogenes cease to grow due to high polymerase expression levels. These colonies can be identified on the original plate, and subsequently picked and characterized.

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