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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVING BIOCHEMICAL PROCESSES FOR THE PRODUCTION OF SUSTAINABLE FUELS AND CHEMICALS

Location: Renewable Product Technology Research Unit

Title: Design and construction of a first-generation high-throughput integrated molecular biology platform for production of optimized synthetic genes and improved industrial strains

Authors
item Hughes, Stephen
item Butt, Tauseed -
item Bartolett, Scott -
item Riedmuller, Steven -
item Sterner, David -
item Farrelly, Philip -
item Bischoff, Kenneth
item Liu, Siqing
item Rich, Joseph

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 8, 2012
Publication Date: March 8, 2012
Citation: Hughes, S.R., Butt, T.R., Bartolett, S., Riedmuller, S., Sterner, D., Farrelly, P., Bischoff, K.M., Liu, S., Rich, J.O. 2012. Design and construction of a first-generation high-throughput integrated molecular biology platform for production of optimized synthetic genes and improved industrial strains [abstract]. CBRD Advisory Board.

Technical Abstract: The molecular biological techniques for plasmid-based assembly and cloning of synthetic assembled gene open reading frames are essential for elucidating the function of the proteins encoded by the genes. These techniques involve the production of full-length cDNA libraries as a source of plasmid-based clones to express the desired protein in active form for determination of its function. These clones can be used in mutagenesis strategies to modify proteins for optimized functions. Plasmid-based functional proteomics requires rapid plasmid preparation methods to obtain adequate quantities of high-quality plasmid DNA to conduct all required steps in the process from creation of plasmid libraries to functional testing of expressed proteins. Because the plasmid libraries are composed of several thousand unique genes, automation of the process is essential. The ideal system would be an automated integrated programmable workcell capable of producing full-length cDNA libraries, colony picking, isolating plasmid DNA, transforming yeast and bacteria, expressing protein, and performing appropriate functional assays. Such an automated system requires the integration of different equipment and instruments with the desired capabilities. It is also possible to generate thousands of variants of one open reading frame. It is also possible to irradiate cells with UV-C light in order to produce cells with multiple mutations. Several examples of workcell paradigms will be used for automated assembly and amino acid scanning mutagenesis of open reading frames as well as automated irradiation of various microbes for trait improvements.

Last Modified: 12/25/2014
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