A high-capacity gene stacking toolkit for the oleaginous microalga, Nannochloropsis oceanica CCMP1779

Poliner, Eric; Clark, Evan; Cummings, Cameron; Benning, Christoph; Farre, Eva M.

doi:10.1016/j.algal.2019.101664

A high-capacity gene stacking toolkit for the oleaginous microalga, Nannochloropsis oceanica CCMP1779

Journal Article · Tue Dec 24 00:00:00 EST 2019 · Algal Research

DOI:https://doi.org/10.1016/j.algal.2019.101664· OSTI ID:1800724

Poliner, Eric ^[1]; Clark, Evan ^[2]; Cummings, Cameron ^[2]; Benning, Christoph ^[2]; Farre, Eva M. ^[2]

Michigan State Univ., East Lansing, MI (United States); OSTI
Michigan State Univ., East Lansing, MI (United States)

Nannochloropsis oceanica CCMP1779 is an oleaginous heterokont microalga that is particularly amenable to synthetic biology approaches. The underlying complex genetic engineering often requires the expression of multiple transgenes (gene stacking). We integrated several techniques into a vector toolkit for multi-gene expression, including: multiple resistance marker genes, bidirectional promoters, and assembly of multiple expression cassettes into a single vector. We developed a series of gateway entry and destination vectors for assembly of two bidirectional promoter expression cassettes, which facilitates combinatorial high-capacity gene stacking. Several endogenous bidirectional promoters were demonstrated to be effective for driving transgene expression, with a dual-luciferase reporter system used to monitor transgene expression under different environmental conditions. The bidirectional promoter between the nitrate reductase and the nitrate transporter genes drives nitrogen source dependent transcription, enabling conditional transgene expression. Lethal concentrations of the antibiotics, Blasticidin, G418, and Nourseothricin, were determined and the corresponding marker genes used for genomic transformation selection.

View Accepted Manuscript (DOE)

Research Organization:: Michigan State Univ., East Lansing, MI (United States)

Sponsoring Organization:: USDOE; USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: FG02-91ER20021

OSTI ID:: 1800724

Alternate ID(s):: OSTI ID: 1780325

Journal Information:: Algal Research, Journal Name: Algal Research Vol. 45; ISSN 2211-9264

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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