title: A method for multiplex gene synthesis employing error correction based on expression.
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.
authors: Department of Bioengineering, University of California, Berkeley, CA, United States of America.
authors: Timothy H-C
authors: TH
authors: Hsiau
authors: Department of Bioengineering, University of California, Berkeley, CA, United States of America.
authors: David
authors: D
authors: Sukovich
authors: Department of Bioengineering, University of California, Berkeley, CA, United States of America.
authors: Phillip
authors: P
authors: Elms
authors: Department of Bioengineering, University of California, Berkeley, CA, United States of America.
authors: Robin N
authors: RN
authors: Prince
authors: Tobias
authors: T
authors: Strittmatter
authors: Department of Bioengineering, University of California, Berkeley, CA, United States of America.
authors: Tobias
authors: T
authors: Stritmatter
authors: Department of Electrical Engineering and Computer Science, University of California, Berkeley, CA, United States of America.
authors: Paul
authors: P
authors: Ruan
authors: Agilent Technologies, Santa Clara, CA, United States of America.
authors: Bo
authors: B
authors: Curry
authors: Agilent Technologies, Santa Clara, CA, United States of America.
authors: Paige
authors: P
authors: Anderson
authors: Agilent Technologies, Santa Clara, CA, United States of America.
authors: Jeff
authors: J
authors: Sampson
authors: Department of Bioengineering, University of California, Berkeley, CA, United States of America; Synthetic Biology Institute, University of California, Berkeley, CA, United States of America.
authors: J Christopher
authors: JC
authors: Anderson
mesh_terms: D004247:DNA / Q000138:chemical synthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005813:Genes, Synthetic; D059014:High-Throughput Nucleotide Sequencing; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides / Q000096:biosynthesis / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics*
chemical_list: D009841:Oligonucleotides; D004247:DNA