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Title: Designer diatom episomes delivered by bacterial conjugation

Abstract

Eukaryotic microalgae hold great promise for the bioproduction of fuels and higher value chemicals. However, compared with model genetic organisms such as Escherichia coli and Saccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement has been hampered by the inefficient genetic tools. To date, many algal species are transformable only via particle bombardment, and the introduced DNA is integrated randomly into the nuclear genome. Here we describe the first nuclear episomal vector for diatoms and a plasmid delivery method via conjugation from Escherichia coli to the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. We identify a yeast-derived sequence that enables stable episome replication in these diatoms even in the absence of antibiotic selection and show that episomes are maintained as closed circles at copy number equivalent to native chromosomes. This highly efficient genetic system facilitates high-throughput functional characterization of algal genes and accelerates molecular phytoplankton research.

Authors:
 [1];  [2];  [3];  [3];  [1];  [1];  [1];  [3];  [4];  [3];  [3];  [3];  [4];  [1];  [1];  [1];  [5];  [2];  [3];  [1]
  1. J. Craig Venter Inst., La Jolla, CA (United States). Synthetic Biology and Bioenergy Group
  2. J. Craig Venter Inst., La Jolla, CA (United States). Microbial and Environmental Genomics Group; Univ. of California, San Diego, CA (United States). Integrative Oceanography Division. Scripps Inst. of Oceanography
  3. J. Craig Venter Inst., La Jolla, CA (United States). Microbial and Environmental Genomics Group
  4. Univ. of California, San Diego, CA (United States). National Center for Microscopy and Imaging Research
  5. J. Craig Venter Inst., La Jolla, CA (United States). Synthetic Biology and Bioenergy Group. Microbial and Environmental Genomics Group
Publication Date:
Research Org.:
J. Craig Venter Inst., La Jolla, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); Synthetic Genomics Inc. (United States); Gordon and Betty Moore Foundation (United States); Natural Sciences and Engineering Research Council of Canada (NSERC)
OSTI Identifier:
1455147
Grant/Contract Number:  
SC0008593; GBMF3828
Resource Type:
Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
Journal Volume: 6; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Karas, Bogumil J., Diner, Rachel E., Lefebvre, Stephane C., McQuaid, Jeff, Phillips, Alex P. R., Noddings, Chari M., Brunson, John K., Valas, Ruben E., Deerinck, Thomas J., Jablanovic, Jelena, Gillard, Jeroen T. F., Beeri, Karen, Ellisman, Mark H., Glass, John I., Hutchison, Clyde A., Smith, Hamilton O., Venter, J. Craig, Allen, Andrew E., Dupont, Christopher L., and Weyman, Philip D. Designer diatom episomes delivered by bacterial conjugation. United States: N. p., 2015. Web. doi:10.1038/ncomms7925.
Karas, Bogumil J., Diner, Rachel E., Lefebvre, Stephane C., McQuaid, Jeff, Phillips, Alex P. R., Noddings, Chari M., Brunson, John K., Valas, Ruben E., Deerinck, Thomas J., Jablanovic, Jelena, Gillard, Jeroen T. F., Beeri, Karen, Ellisman, Mark H., Glass, John I., Hutchison, Clyde A., Smith, Hamilton O., Venter, J. Craig, Allen, Andrew E., Dupont, Christopher L., & Weyman, Philip D. Designer diatom episomes delivered by bacterial conjugation. United States. doi:10.1038/ncomms7925.
Karas, Bogumil J., Diner, Rachel E., Lefebvre, Stephane C., McQuaid, Jeff, Phillips, Alex P. R., Noddings, Chari M., Brunson, John K., Valas, Ruben E., Deerinck, Thomas J., Jablanovic, Jelena, Gillard, Jeroen T. F., Beeri, Karen, Ellisman, Mark H., Glass, John I., Hutchison, Clyde A., Smith, Hamilton O., Venter, J. Craig, Allen, Andrew E., Dupont, Christopher L., and Weyman, Philip D. Tue . "Designer diatom episomes delivered by bacterial conjugation". United States. doi:10.1038/ncomms7925. https://www.osti.gov/servlets/purl/1455147.
@article{osti_1455147,
title = {Designer diatom episomes delivered by bacterial conjugation},
author = {Karas, Bogumil J. and Diner, Rachel E. and Lefebvre, Stephane C. and McQuaid, Jeff and Phillips, Alex P. R. and Noddings, Chari M. and Brunson, John K. and Valas, Ruben E. and Deerinck, Thomas J. and Jablanovic, Jelena and Gillard, Jeroen T. F. and Beeri, Karen and Ellisman, Mark H. and Glass, John I. and Hutchison, Clyde A. and Smith, Hamilton O. and Venter, J. Craig and Allen, Andrew E. and Dupont, Christopher L. and Weyman, Philip D.},
abstractNote = {Eukaryotic microalgae hold great promise for the bioproduction of fuels and higher value chemicals. However, compared with model genetic organisms such as Escherichia coli and Saccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement has been hampered by the inefficient genetic tools. To date, many algal species are transformable only via particle bombardment, and the introduced DNA is integrated randomly into the nuclear genome. Here we describe the first nuclear episomal vector for diatoms and a plasmid delivery method via conjugation from Escherichia coli to the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. We identify a yeast-derived sequence that enables stable episome replication in these diatoms even in the absence of antibiotic selection and show that episomes are maintained as closed circles at copy number equivalent to native chromosomes. This highly efficient genetic system facilitates high-throughput functional characterization of algal genes and accelerates molecular phytoplankton research.},
doi = {10.1038/ncomms7925},
journal = {Nature Communications},
number = ,
volume = 6,
place = {United States},
year = {2015},
month = {4}
}

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Works referenced in this record:

Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome
journal, May 2010


Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009

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