Abstract
Testing the many hypotheses from genomics and systems biology experiments demands accurate and cost-effective gene and genome synthesis. Here we describe a microchip-based technology for multiplex gene synthesis. Pools of thousands of 'construction' oligonucleotides and tagged complementary 'selection' oligonucleotides are synthesized on photo-programmable microfluidic chips, released, amplified and selected by hybridization to reduce synthesis errors ninefold. A one-step polymerase assembly multiplexing reaction assembles these into multiple genes. This technology enabled us to synthesize all 21 genes that encode the proteins of the Escherichia coli 30S ribosomal subunit, and to optimize their translation efficiency in vitro through alteration of codon bias. This is a significant step towards the synthesis of ribosomes in vitro and should have utility for synthetic biology in general.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Escherichia coli / genetics
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Escherichia coli Proteins / biosynthesis
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Escherichia coli Proteins / genetics
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Genes / genetics*
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Genomics / economics
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Genomics / instrumentation*
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Genomics / methods*
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Microfluidics / economics
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Microfluidics / instrumentation
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Microfluidics / methods
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Molecular Sequence Data
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Nucleic Acid Hybridization
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Oligonucleotide Array Sequence Analysis / economics
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Oligonucleotide Array Sequence Analysis / instrumentation*
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Oligonucleotides / biosynthesis
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Oligonucleotides / genetics
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Oligonucleotides / metabolism
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Protein Biosynthesis / genetics*
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Protein Subunits / genetics
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Research Design
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Ribosomal Proteins / biosynthesis
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Ribosomal Proteins / genetics
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Ribosomes / genetics
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Sensitivity and Specificity
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Software*
Substances
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Escherichia coli Proteins
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Oligonucleotides
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Protein Subunits
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Ribosomal Proteins