Assembly of large, high G+C bacterial DNA fragments in yeast

Vladimir N. Noskov; Bogumil J. Karas; Lei Young; Ray Yuan Chuang; Daniel G. Gibson; Ying Chi Lin; Jason Stam; Isaac T. Yonemoto; Yo Suzuki; Cynthia Andrews-Pfannkoch; John I. Glass; Hamilton O. Smith; Clyde A. Hutchison; J. Craig Venter; Philip D. Weyman

doi:10.1021/sb3000194

Assembly of large, high G+C bacterial DNA fragments in yeast

Vladimir N. Noskov, Bogumil J. Karas, Lei Young, Ray Yuan Chuang, Daniel G. Gibson, Ying Chi Lin, Jason Stam, Isaac T. Yonemoto, Yo Suzuki, Cynthia Andrews-Pfannkoch, John I. Glass, Hamilton O. Smith, Clyde A. Hutchison, J. Craig Venter, Philip D. Weyman

Research output: Contribution to journal › Article › peer-review

Abstract

The ability to assemble large pieces of prokaryotic DNA by yeast recombination has great application in synthetic biology, but cloning large pieces of high G+C prokaryotic DNA in yeast can be challenging. Additional considerations in cloning large pieces of high G+C DNA in yeast may be related to toxic genes, to the size of the DNA, or to the absence of yeast origins of replication within the sequence. As an example of our ability to clone high G+C DNA in yeast, we chose to work with Synechococcus elongatus PCC 7942, which has an average G+C content of 55%. We determined that no regions of the chromosome are toxic to yeast and that S. elongatus DNA fragments over ̃200 kb are not stably maintained. DNA constructs with a total size under 200 kb could be readily assembled, even with 62 kb of overlapping sequence between pieces. Addition of yeast origins of replication throughout allowed us to increase the total size of DNA that could be assembled to at least 454 kb. Thus, cloning strategies utilizing yeast recombination with large, high G+C prokaryotic sequences should include yeast origins of replication as a part of the design process.

Original language	English (US)
Pages (from-to)	267-273
Number of pages	7
Journal	ACS Synthetic Biology
Volume	1
Issue number	7
DOIs	https://doi.org/10.1021/sb3000194
State	Published - Jul 20 2012
Externally published	Yes

Keywords

Cyanobacteria
DNA assembly
Synechococcus
Transformation-associated recombination
Yeast

ASJC Scopus subject areas

Biomedical Engineering
Biochemistry, Genetics and Molecular Biology (miscellaneous)

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10.1021/sb3000194

Cite this

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title = "Assembly of large, high G+C bacterial DNA fragments in yeast",

abstract = "The ability to assemble large pieces of prokaryotic DNA by yeast recombination has great application in synthetic biology, but cloning large pieces of high G+C prokaryotic DNA in yeast can be challenging. Additional considerations in cloning large pieces of high G+C DNA in yeast may be related to toxic genes, to the size of the DNA, or to the absence of yeast origins of replication within the sequence. As an example of our ability to clone high G+C DNA in yeast, we chose to work with Synechococcus elongatus PCC 7942, which has an average G+C content of 55%. We determined that no regions of the chromosome are toxic to yeast and that S. elongatus DNA fragments over{\~ }200 kb are not stably maintained. DNA constructs with a total size under 200 kb could be readily assembled, even with 62 kb of overlapping sequence between pieces. Addition of yeast origins of replication throughout allowed us to increase the total size of DNA that could be assembled to at least 454 kb. Thus, cloning strategies utilizing yeast recombination with large, high G+C prokaryotic sequences should include yeast origins of replication as a part of the design process.",

keywords = "Cyanobacteria, DNA assembly, Synechococcus, Transformation-associated recombination, Yeast",

author = "Noskov, {Vladimir N.} and Karas, {Bogumil J.} and Lei Young and Chuang, {Ray Yuan} and Gibson, {Daniel G.} and Lin, {Ying Chi} and Jason Stam and Yonemoto, {Isaac T.} and Yo Suzuki and Cynthia Andrews-Pfannkoch and Glass, {John I.} and Smith, {Hamilton O.} and Hutchison, {Clyde A.} and Venter, {J. Craig} and Weyman, {Philip D.}",

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T1 - Assembly of large, high G+C bacterial DNA fragments in yeast

AU - Noskov, Vladimir N.

AU - Karas, Bogumil J.

AU - Young, Lei

AU - Chuang, Ray Yuan

AU - Gibson, Daniel G.

AU - Lin, Ying Chi

AU - Stam, Jason

AU - Yonemoto, Isaac T.

AU - Suzuki, Yo

AU - Andrews-Pfannkoch, Cynthia

AU - Glass, John I.

AU - Smith, Hamilton O.

AU - Hutchison, Clyde A.

AU - Venter, J. Craig

AU - Weyman, Philip D.

PY - 2012/7/20

Y1 - 2012/7/20

N2 - The ability to assemble large pieces of prokaryotic DNA by yeast recombination has great application in synthetic biology, but cloning large pieces of high G+C prokaryotic DNA in yeast can be challenging. Additional considerations in cloning large pieces of high G+C DNA in yeast may be related to toxic genes, to the size of the DNA, or to the absence of yeast origins of replication within the sequence. As an example of our ability to clone high G+C DNA in yeast, we chose to work with Synechococcus elongatus PCC 7942, which has an average G+C content of 55%. We determined that no regions of the chromosome are toxic to yeast and that S. elongatus DNA fragments over ̃200 kb are not stably maintained. DNA constructs with a total size under 200 kb could be readily assembled, even with 62 kb of overlapping sequence between pieces. Addition of yeast origins of replication throughout allowed us to increase the total size of DNA that could be assembled to at least 454 kb. Thus, cloning strategies utilizing yeast recombination with large, high G+C prokaryotic sequences should include yeast origins of replication as a part of the design process.

AB - The ability to assemble large pieces of prokaryotic DNA by yeast recombination has great application in synthetic biology, but cloning large pieces of high G+C prokaryotic DNA in yeast can be challenging. Additional considerations in cloning large pieces of high G+C DNA in yeast may be related to toxic genes, to the size of the DNA, or to the absence of yeast origins of replication within the sequence. As an example of our ability to clone high G+C DNA in yeast, we chose to work with Synechococcus elongatus PCC 7942, which has an average G+C content of 55%. We determined that no regions of the chromosome are toxic to yeast and that S. elongatus DNA fragments over ̃200 kb are not stably maintained. DNA constructs with a total size under 200 kb could be readily assembled, even with 62 kb of overlapping sequence between pieces. Addition of yeast origins of replication throughout allowed us to increase the total size of DNA that could be assembled to at least 454 kb. Thus, cloning strategies utilizing yeast recombination with large, high G+C prokaryotic sequences should include yeast origins of replication as a part of the design process.

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Assembly of large, high G+C bacterial DNA fragments in yeast

Abstract

Keywords

ASJC Scopus subject areas

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