Presentation Title

Developing healthier Roundup-resistant cassava using deep mutational scanning

Start Date

November 2016

End Date

November 2016

Location

HUB 302-47

Type of Presentation

Poster

Abstract

Glyphosate is a commonly-used broad spectrum herbicide and the active ingredient in Roundup. Glyphosate inhibits EPSP synthase (EPSPs), an enzyme needed to produce three aromatic amino acids. By inhibiting production of these three amino acids, glyphosate inhibits plant growth, eventually causing plant death. The purpose of this project is to develop a glyphosate-resistant variety of cassava, an important staple crop in sub-Saharan Africa. Cassava farmers are often constrained by weed proliferation, as more than 50% of cassava production costs is allocated to weeding. Glyphosate resistance typically occurs from mutations (Thr181Ile and Pro185Ser181 in cassava), but these cause slow-growing plants. Our goal is to identify a mutation(s) in the gene encoding EPSP synthase that confers resistance to glyphosate but does not inhibit normal enzyme function. We created a library of EPSPs variants by randomizing the nucleotides for amino acids 181 and 185 and introduced the EPSPs variants into E. coli lacking its native EPSPs gene. We grew a library of E. coli containing these EPSPs mutants in media lacking the amino acids EPSPs synthesizes and supplemented with glyphosate, and collected the surviving colonies. 190 colonies were individually assessed for glyphosate tolerance by measuring their growth rate in 50mM glyphosate. The 20 colonies that grew the best were sequenced. The same 190 colonies were pooled together and sequenced in a high throughput manner. The proportion of reads for each variant can be related to the proportion of colonies in the culture carrying each variant. Wild type cassava EPSPs and a double mutant, Thr181Ile and Pro185Ala, were the most abundant.

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Nov 12th, 1:00 PM Nov 12th, 2:00 PM

Developing healthier Roundup-resistant cassava using deep mutational scanning

HUB 302-47

Glyphosate is a commonly-used broad spectrum herbicide and the active ingredient in Roundup. Glyphosate inhibits EPSP synthase (EPSPs), an enzyme needed to produce three aromatic amino acids. By inhibiting production of these three amino acids, glyphosate inhibits plant growth, eventually causing plant death. The purpose of this project is to develop a glyphosate-resistant variety of cassava, an important staple crop in sub-Saharan Africa. Cassava farmers are often constrained by weed proliferation, as more than 50% of cassava production costs is allocated to weeding. Glyphosate resistance typically occurs from mutations (Thr181Ile and Pro185Ser181 in cassava), but these cause slow-growing plants. Our goal is to identify a mutation(s) in the gene encoding EPSP synthase that confers resistance to glyphosate but does not inhibit normal enzyme function. We created a library of EPSPs variants by randomizing the nucleotides for amino acids 181 and 185 and introduced the EPSPs variants into E. coli lacking its native EPSPs gene. We grew a library of E. coli containing these EPSPs mutants in media lacking the amino acids EPSPs synthesizes and supplemented with glyphosate, and collected the surviving colonies. 190 colonies were individually assessed for glyphosate tolerance by measuring their growth rate in 50mM glyphosate. The 20 colonies that grew the best were sequenced. The same 190 colonies were pooled together and sequenced in a high throughput manner. The proportion of reads for each variant can be related to the proportion of colonies in the culture carrying each variant. Wild type cassava EPSPs and a double mutant, Thr181Ile and Pro185Ala, were the most abundant.