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A method for multiplex gene synthesis employing error correction based on expression.

Timothy H-C Hsiau1, David Sukovich1, Phillip Elms1

  • 1Department of Bioengineering, University of California, Berkeley, CA, United States of America.

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|March 20, 2015
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Summary
This summary is machine-generated.

We developed a new method, Multiplex Library Synthesis and Expression Correction (MuLSEC), to rapidly characterize many proteins. This technique lowers costs and improves efficiency for engineering organisms with new genetic circuits.

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Engineering organisms with novel biosynthetic pathways and genetic circuits is hindered by limited protein characterization data and high synthetic DNA costs.
  • Advancements in DNA synthesis and sequencing offer opportunities for scalable, cost-effective assays to determine protein biochemical characteristics.

Purpose of the Study:

  • To develop a method for rapid, cost-effective assembly, error correction, and expression characterization of large gene libraries.
  • To overcome limitations in protein characterization for synthetic biology applications.

Main Methods:

  • Developed the Multiplex Library Synthesis and Expression Correction (MuLSEC) method for one-pot gene library assembly from microarray-synthesized oligonucleotides.
  • Utilized an ampicillin-based quality control selection for error correction and to select for properly expressed and folded proteins in E. coli.
  • Employed next-generation sequencing for quantitative analysis of gene expression characteristics.

Main Results:

  • Successfully demonstrated the feasibility of MuLSEC by building and testing over 90 genes for soluble expression.
  • The MuLSEC method enables multiplex gene synthesis and characterization, reducing reliance on robotic liquid handling.
  • Achieved quantitative analysis of gene expression characteristics through deep sequencing of post-selection DNA.

Conclusions:

  • MuLSEC significantly enhances the efficiency and reduces the cost of protein characterization for synthetic biology.
  • This methodology streamlines the process of mining biochemical protein characteristics, facilitating the engineering of novel biological functions.
  • The approach leverages scalable technologies like multiplex oligonucleotide synthesis and deep sequencing, poised for further cost reduction.