Error correction of microchip synthesized genes using Surveyor nuclease

The development of economical and high-throughput gene synthesis technology has been hampered by the high occurrence of errors in the synthesized products, which requires expensive labor and time to correct. Here, we describe an error correction reaction (ECR), which employs Surveyor, a mismatch-specific DNA endonuclease, to remove errors from synthetic genes. In ECR reactions, errors are revealed as mismatches by re-annealing of the synthetic gene products. Mismatches are recognized and excised by a combination of mismatch-specific endonuclease and 3′→5′ exonuclease activities in the reaction mixture. Finally, overlap extension polymerase chain reaction (OE-PCR) re-assembles the resulting fragments into intact genes. The process can be iterated for increased fidelity. With two iterations, we were able to reduce errors in synthetic genes by >16-fold, yielding a final error rate of ∼1 in 8700 bp

Enabling Technologies for Synthetic Biology: Gene Synthesis and Error-Correction from a Microarray-Microfluidic Integrated Device

<p>Promising applications in the design of various biological systems hold critical implications as heralded in the rising field of synthetic biology. But, to achieve these goals, the ability to synthesize in situ DNA constructs of any size or sequence rapidly, accurately and economically is crucial. Today, the process of DNA oligonucleotide synthesis has been automated but the overall development of gene and genome synthesis technology has far lagged behind that of gene and genome sequencing. This has meant that numerous ideas go unfulfilled due to scale, cost and impediments in the quality of DNA due to synthesis errors. </p><p>This thesis presents the development of a multi-tool ensemble platform targeted at gene synthesis. An inkjet oligonucleotide synthesizer is constructed to synthesize DNA microarrays onto silica functionalized cylic olefin copolymer substrates. The arrays are married to microfluidic wells that provide a chamber to for enzymatic amplification and assembly of the DNA from the microarrays into a larger construct. Harvested product is then amplified off-chip and error corrected using a mismatch endonuclease-based reaction. This platform has the potential to be particularly low-cost since it employs standard phosphoramidite reagents and parts that are cheaper than optical and electrochemical systems. Genes sized 160 bp to 993 bp were successfully harvested and, after error correction, achieved up to 94% of intended functionality.</p>Dissertatio

Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae

The radical S-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Saccharomyces cerevisiae. Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted nif genes (nifUSXB and fdxN) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including nifUSX and fdxN, and a library consisting of 28 different nifB genes mined from the Structure–Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from Azotobacter vinelandii with NifB from the archaea Methanocaldococcus infernus or Methanothermobacter thermautotrophicus yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured S. cerevisiae coexpressing M. thermautotrophicus NifB with A. vinelandii NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism.United States. Defense Advanced Research Projects Agency. Living Foundries Program ( Award HR0011-15-C-0084

One-Pot Assembly of a Hetero-dimeric DNA Origami from Chip-Derived Staples and Double-Stranded Scaffold

Although structural DNA nanotechnology, and especially scaffolded DNA origami, hold great promise for bottom-up fabrication of novel nanoscale materials and devices, concerns about scalability have tempered widespread enthusiasm. Here we report a single-pot reaction where both strands of double-stranded M13-bacteriophage DNA are simultaneously folded into two distinct shapes that then heterodimerize with high yield. The fully addressable, two-dimensional heterodimer DNA origami, with twice the surface area of standard M13 origami, formed in high yield (81% of the well-formed monomers undergo dimerization). We also report the concurrent production of entire sets of staple strands by a unique, nicking strand-displacement amplification (nSDA) involving reusable surface-bound template strands that were synthesized <i>in situ</i> using a custom piezoelectric inkjet system. The combination of chip-based staple strand production, double-sized origami, and high-yield one-pot assembly markedly increases the useful scale of DNA origami

PCR amplification of oligonucleotides with wide range GC contents with subcycling and different additives.

<p>A. No additives; B. 60% deaza-dGTP; C. 0.2M betaine. First lane on the left of each gel: MW markers. Lanes numbered indicates oligonucleotides of varying GC content as follows: 1) 10% GC; 2) 21%GC; 3) 33%GC; 4) 44%GC; 5) 56%GC; 6) 67%GC; 7) 79% GC; 8) 90%GC. Results are based on electronic gels created by electrophoresis using a Perkin Elmer GX instrument with a 5k chip. *Bin shows expected PCR pattern where a strong 200bp product band is seen. PCR reactions were not purified and primers can be seen at the bottom of each sample.</p

Percent of successful builds of DNA constructs with varying GC content using the standard protocol.

<p>The same data is visualized as a bar graph in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156478#pone.0156478.g005" target="_blank">Fig 5</a>.</p

Percent of successful builds of DNA constructs with varying GC content.

<p>The bar graph is a visual representation of the data in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156478#pone.0156478.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156478#pone.0156478.t002" target="_blank">2</a>. Blue bars—represent the successful builds with the standard protocol. Red bars—represent the successful builds with the broad spectrum protocol.</p