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Title: Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling

Abstract

The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here in this paper we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ~10%of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to designmore » and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.« less

Authors:
 [1];  [2];  [1];  [2];  [3];  [1];  [1];  [2];  [1];  [1];  [1];  [4];  [1];  [2];  [2];  [5];  [1];  [1];  [1];  [6] more »;  [6] « less
  1. J. Craig Venter Inst., La Jolla, CA (United States). Synthetic Biology Group
  2. J. Craig Venter Inst., Rockville, MD (United States). Synthetic Biology Group
  3. J. Craig Venter Inst., La Jolla, CA (United States). Synthetic Biology Group; Univ. of Missouri, Columbia, MO (United States). Dept. of Molecular Microbiology and Immunology
  4. J. Craig Venter Inst., La Jolla, CA (United States). Synthetic Biology Group; Univ. of California, San Diego, CA (United States). School of Medicine
  5. Univ. of California, La Jolla, CA (United States). School of Medicine
  6. J. Craig Venter Inst., La Jolla, CA (United States). Synthetic Biology Group; J. Craig Venter Inst., Rockville, MD (United States). Synthetic Biology Group
Publication Date:
Research Org.:
J. Craig Venter Inst., La Jolla, CA (United States)
Sponsoring Org.:
USDOE; US Defense Advanced Research Projects Agency (DARPA)
OSTI Identifier:
1344872
Grant/Contract Number:  
FC02-02ER63453; EE0006109; N66001-12-C-4039
Resource Type:
Accepted Manuscript
Journal Name:
Genome Research
Additional Journal Information:
Journal Volume: 25; Journal Issue: 3; Journal ID: ISSN 1088-9051
Publisher:
Cold Spring Harbor Laboratory Press
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Suzuki, Yo, Assad-Garcia, Nacyra, Kostylev, Maxim, Noskov, Vladimir N., Wise, Kim S., Karas, Bogumil J., Stam, Jason, Montague, Michael G., Hanly, Timothy J., Enriquez, Nico J., Ramon, Adi, Goldgof, Gregory M., Richter, R. Alexander, Vashee, Sanjay, Chuang, Ray-Yuan, Winzeler, Elizabeth A., Hutchison, Clyde A., Gibson, Daniel G., Smith, Hamilton O., Glass, John I., and Venter, J. Craig. Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling. United States: N. p., 2015. Web. doi:10.1101/gr.182477.114.
Suzuki, Yo, Assad-Garcia, Nacyra, Kostylev, Maxim, Noskov, Vladimir N., Wise, Kim S., Karas, Bogumil J., Stam, Jason, Montague, Michael G., Hanly, Timothy J., Enriquez, Nico J., Ramon, Adi, Goldgof, Gregory M., Richter, R. Alexander, Vashee, Sanjay, Chuang, Ray-Yuan, Winzeler, Elizabeth A., Hutchison, Clyde A., Gibson, Daniel G., Smith, Hamilton O., Glass, John I., & Venter, J. Craig. Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling. United States. https://doi.org/10.1101/gr.182477.114
Suzuki, Yo, Assad-Garcia, Nacyra, Kostylev, Maxim, Noskov, Vladimir N., Wise, Kim S., Karas, Bogumil J., Stam, Jason, Montague, Michael G., Hanly, Timothy J., Enriquez, Nico J., Ramon, Adi, Goldgof, Gregory M., Richter, R. Alexander, Vashee, Sanjay, Chuang, Ray-Yuan, Winzeler, Elizabeth A., Hutchison, Clyde A., Gibson, Daniel G., Smith, Hamilton O., Glass, John I., and Venter, J. Craig. Thu . "Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling". United States. https://doi.org/10.1101/gr.182477.114. https://www.osti.gov/servlets/purl/1344872.
@article{osti_1344872,
title = {Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling},
author = {Suzuki, Yo and Assad-Garcia, Nacyra and Kostylev, Maxim and Noskov, Vladimir N. and Wise, Kim S. and Karas, Bogumil J. and Stam, Jason and Montague, Michael G. and Hanly, Timothy J. and Enriquez, Nico J. and Ramon, Adi and Goldgof, Gregory M. and Richter, R. Alexander and Vashee, Sanjay and Chuang, Ray-Yuan and Winzeler, Elizabeth A. and Hutchison, Clyde A. and Gibson, Daniel G. and Smith, Hamilton O. and Glass, John I. and Venter, J. Craig},
abstractNote = {The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here in this paper we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ~10%of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.},
doi = {10.1101/gr.182477.114},
journal = {Genome Research},
number = 3,
volume = 25,
place = {United States},
year = {2015},
month = {2}
}

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