Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries.

BMC Biotechnology
Thomas C ScanlonKarl E Griswold

Abstract

In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. In order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by DNA sequencing. It was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on a...Continue Reading

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Citations

Jul 8, 2010·ACS Chemical Biology·Thomas C ScanlonKarl E Griswold
Jun 23, 2012·Nature Protocols·Ryan HietpasDaniel N A Bolon
Feb 5, 2013·Journal of Molecular Biology·Benjamin P RoscoeDaniel N A Bolon
Aug 21, 2013·Biotechnology and Bioengineering·Thomas C ScanlonKarl E Griswold
Jan 22, 2015·ACS Chemical Biology·Sarah M DostalKarl E Griswold
Mar 19, 2014·Bioengineered·Karl E GriswoldLaurie W Leclair
Feb 18, 2011·Bioengineered Bugs·Thomas C ScanlonKarl E Griswold
Mar 5, 2015·FEMS Yeast Research·Chia-Ching ChouMarc R Gartenberg
Sep 14, 2017·Nature·Tyler N StarrJoseph W Thornton

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Methods Mentioned

BETA
two hybrid
PCR
amplicon sequencing
electrophoresis

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