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

PLoS One. 2015 Mar 19;10(3):e0119927. doi: 10.1371/journal.pone.0119927. eCollection 2015.

Abstract

Our ability to engineer organisms with new biosynthetic pathways and genetic circuits is limited by the availability of protein characterization data and the cost of synthetic DNA. With new tools for reading and writing DNA, there are opportunities for scalable assays that more efficiently and cost effectively mine for biochemical protein characteristics. To that end, we have developed the Multiplex Library Synthesis and Expression Correction (MuLSEC) method for rapid assembly, error correction, and expression characterization of many genes as a pooled library. This methodology enables gene synthesis from microarray-synthesized oligonucleotide pools with a one-pot technique, eliminating the need for robotic liquid handling. Post assembly, the gene library is subjected to an ampicillin based quality control selection, which serves as both an error correction step and a selection for proteins that are properly expressed and folded in E. coli. Next generation sequencing of post selection DNA enables quantitative analysis of gene expression characteristics. We demonstrate the feasibility of this approach by building and testing over 90 genes for empirical evidence of soluble expression. This technique reduces the problem of part characterization to multiplex oligonucleotide synthesis and deep sequencing, two technologies under extensive development with projected cost reduction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA / chemical synthesis
  • DNA / genetics*
  • Escherichia coli / genetics
  • Gene Expression Regulation
  • Genes, Synthetic*
  • High-Throughput Nucleotide Sequencing
  • Oligonucleotide Array Sequence Analysis
  • Oligonucleotides / biosynthesis
  • Oligonucleotides / genetics*
  • Protein Biosynthesis / genetics*

Substances

  • Oligonucleotides
  • DNA

Grants and funding

Work was supported by funding from Agilent Technologies via the Synthetic Biology Institute. Agilent provided some of the software development. Additional software and wetlab experiments were performed at UC Berkeley. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.