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

PloS One
Timothy H-C HsiauJ Christopher Anderson

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 ch...Continue Reading

References

Jul 15, 1999·Nature Biotechnology·G S WaldoT C Terwilliger
Sep 3, 1999·Nucleic Acids Research·D A ShaginM V Matz
Dec 8, 2004·Nature Biotechnology·Stéphanie CabantousGeoffrey S Waldo
Feb 3, 2006·Protein Science : a Publication of the Protein Society·Adam C FisherMatthew P DeLisa
Jun 23, 2006·Nature Methods·Richard WilliamsAndrew D Griffiths
Jun 12, 2008·PloS One·Stéphanie CabantousGeoffrey S Waldo
Jun 21, 2008·The Journal of Immunology : Official Journal of the American Association of Immunologists·Takehiko UeyamaNaoaki Saito
Jan 28, 2009·Proceedings of the National Academy of Sciences of the United States of America·Qikai XuStephen J Elledge
Oct 16, 2009·Protein Science : a Publication of the Protein Society·Hyung-Kwon LimMatthew P DeLisa
Dec 17, 2009·Molecular Cell·Linda FoitJames C A Bardwell
Nov 30, 2010·Nature Biotechnology·Sriram KosuriGeorge M Church
Apr 26, 2011·Nature Biotechnology·Jiayuan QuanJingdong Tian
Jun 7, 2011·Proceedings of the National Academy of Sciences of the United States of America·Julius B LucksAdam P Arkin
Jul 27, 2011·Journal of Biological Engineering·Joshua T KittlesonJ Christopher Anderson
Jun 19, 2012·Nucleic Acids Research·Hwangbeom KimDuhee Bang
Jul 24, 2012·Current Opinion in Chemical Biology·Joshua T KittlesonJ Christopher Anderson
Jul 31, 2013·Molecular & Cellular Proteomics : MCP·Ikjin KimStanley Fields
Aug 9, 2013·Proceedings of the National Academy of Sciences of the United States of America·Sriram KosuriGeorge M Church
Nov 21, 2013·Nucleic Acids Research·UNKNOWN UniProt Consortium
May 16, 2014·Nucleic Acids Research·Wen WanJiong Hong

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Citations

Jul 22, 2020·Nucleic Acids Research·Angus M SidoreSriram Kosuri

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Methods Mentioned

BETA
protein folding
PCR
chip
electrophoresis
Assay
PCRs
fluorescence-activated cell sorting

Software Mentioned

Adobe
BRENDA
ImageJ
Photoshop
MuLSEC
Samtools
Excel
BWA

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