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.

Organism Engineering for the Bioproduction of the Triaminotrinitrobenzene (TATB) Precursor Phloroglucinol (PG).

Organism engineering requires the selection of an appropriate chassis, editing its genome, combining traits from different source species, and controlling genes with synthetic circuits. When a strain is needed for a new target objective, for example, to produce a chemical-of-need, the best strains, genes, techniques, software, and expertise may be distributed across laboratories. Here, we report a project where we were assigned phloroglucinol (PG) as a target, and then combined unique capabilities across the United States Army, Navy, and Air Force service laboratories with the shared goal of designing an organism to produce this molecule. In addition to the laboratory strain Escherichia coli, organisms were screened from soil and seawater. Putative PG-producing enzymes were mined from a strain bank of bacteria isolated from aircraft and fuel depots. The best enzyme was introduced into the ocean strain Marinobacter atlanticus CP1 with its genome edited to redirect carbon flux from natural fatty acid ester (FAE) production. PG production was also attempted in Bacillus subtilis and Clostridium acetobutylicum. A genetic circuit was constructed in E. coli that responds to PG accumulation, which was then ported to an in vitro paper-based system that could serve as a platform for future low-cost strain screening or for in-field sensing. Collectively, these efforts show how distributed biotechnology laboratories with domain-specific expertise can be marshalled to quickly provide a solution for a targeted organism engineering project, and highlights data and material sharing protocols needed to accelerate future efforts.

Improved PCR Amplification of Broad Spectrum GC DNA Templates.

Many applications in molecular biology can benefit from improved PCR amplification of DNA segments containing a wide range of GC content. Conventional PCR amplification of DNA sequences with regions of GC less than 30%, or higher than 70%, is complex due to secondary structures that block the DNA polymerase as well as mispriming and mis-annealing of the DNA. This complexity will often generate incomplete or nonspecific products that hamper downstream applications. In this study, we address multiplexed PCR amplification of DNA segments containing a wide range of GC content. In order to mitigate amplification complications due to high or low GC regions, we tested a combination of different PCR cycling conditions and chemical additives. To assess the fate of specific oligonucleotide (oligo) species with varying GC content in a multiplexed PCR, we developed a novel method of sequence analysis. Here we show that subcycling during the amplification process significantly improved amplification of short template pools (~200 bp), particularly when the template contained a low percent of GC. Furthermore, the combination of subcycling and 7-deaza-dGTP achieved efficient amplification of short templates ranging from 10-90% GC composition. Moreover, we found that 7-deaza-dGTP improved the amplification of longer products (~1000 bp). These methods provide an updated approach for PCR amplification of DNA segments containing a broad range of GC content.

Toward larger DNA origami.

Structural DNA nanotechnology, and specifically scaffolded DNA origami, is rapidly developing as a versatile method for bottom-up fabrication of novel nanometer-scale materials and devices. However, lengths of conventional single-stranded scaffolds, for example, 7,249-nucleotide circular genomic DNA from the M13mp18 phage, limit the scales of these uniquely addressable structures. Additionally, increasing DNA origami size generates the cost burden of increased staple-strand synthesis. We addressed this 2-fold problem by developing the following methods: (1) production of the largest to-date biologically derived single-stranded scaffold using a λ/M13 hybrid virus to produce a 51 466-nucleotide DNA in a circular, single-stranded form and (2) inexpensive DNA synthesis via an inkjet-printing process on a chip embossed with functionalized micropillars made from cyclic olefin copolymer. We have experimentally demonstrated very efficient assembly of a 51-kilobasepair origami from the λ/M13 hybrid scaffold folded by chip-derived staple strands. In addition, we have demonstrated two-dimensional, asymmetric origami sheets with controlled global curvature such that they land on a substrate in predictable orientations that have been verified by atomic force microscopy.

Personalized diagnostics and biosensors: a review of the biology and technology needed for personalized medicine.

Exploiting the burgeoning fields of genomics, proteomics and metabolomics improves understanding of human physiology and, critically, the mutations that signal disease susceptibility. Through these emerging fields, rational design approaches to diagnosis, drug development and ultimately personalized medicine are possible. Personalized medicine and point-of-care testing techniques must fulfill a host of constraints for real-world applicability. Point-of-care devices (POCDs) must ultimately provide a cost-effective alternative to expensive and time-consuming laboratory tests in order to assist health care personnel with disease diagnosis and treatment decisions. Sensor technologies are also expanding beyond the more traditional classes of biomarkers--nucleic acids and proteins--to metabolites and direct detection of pathogens, ultimately increasing the palette of available techniques for the use of personalized medicine. The technologies needed to perform such diagnostics have also been rapidly evolving, with each generation being increasingly sensitive and selective while being more resource conscious. Ultimately, the final hurdle for all such technologies is to be able to drive consumer adoption and achieve a meaningful medical outcome for the patient.

Overview of DNA origami for molecular self-assembly.

Judging by the number of atoms and the precision with which they are organized in three-dimensional space, DNA origami assemblies represent the current acme of human molecular engineering accomplishments. A subset of structural DNA nanotechnology, DNA origami makes use of the programmable molecular recognition of complementary DNA cohesions to assemble designed structures. This review will discuss the development of concepts and methods involved in DNA origami with an eye toward future increases in origami size and sequence complexity, as well as exploring different methods for the production of the DNA molecular components (long biologically synthesized scaffold strands and the complex set of chemically synthesized staple strands). In future applications, the incorporation and organization of other materials (metals and other inorganics, protein enzymes, and other nanomaterials) upon or within DNA origami should result in tools for "bottom-up" nanofabrication of biomedical, electronic, and photonic devices and materials.

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 in situ 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.

Error correction in gene synthesis technology.

Accurate, economical and high-throughput gene and genome synthesis is essential to the development of synthetic biology and biotechnology. New large-scale gene synthesis methods harnessing the power of DNA microchips have recently been demonstrated. Yet, the technology is still compromised by a high occurrence of errors in the synthesized products. These errors still require substantial effort to correct. To solve this bottleneck, novel approaches based on new chemistry, enzymology or next generation sequencing have emerged. This review discusses these new trends and promising strategies of error filtration, correction and prevention in de novo gene and genome synthesis. Continued innovation in error correction technologies will enable affordable and large-scale gene and genome synthesis in the near future.

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.

Parallel on-chip gene synthesis and application to optimization of protein expression.

Low-cost, high-throughput gene synthesis and precise control of protein expression are of critical importance to synthetic biology and biotechnology. Here we describe the development of an on-chip gene synthesis technology, which integrates on a single microchip the synthesis of DNA oligonucleotides using inkjet printing, isothermal oligonucleotide amplification and parallel gene assembly. Use of a mismatch-specific endonuclease for error correction results in an error rate of ~0.19 errors per kb. We applied this approach to synthesize pools of thousands of codon-usage variants of lacZα and 74 challenging Drosophila protein antigens, which were then screened for expression in Escherichia coli. In one round of synthesis and screening, we obtained DNA sequences that were expressed at a wide range of levels, from zero to almost 60% of the total cell protein mass. This technology may facilitate systematic investigation of the molecular mechanisms of protein translation and the design, construction and evolution of macromolecular machines, metabolic networks and synthetic cells.

In situ synthesis of DNA microarray on functionalized cyclic olefin copolymer substrate.

Thermoplastic materials such as cyclic-olefin copolymers (COC) provide a versatile and cost-effective alternative to the traditional glass or silicon substrate for rapid prototyping and industrial scale fabrication of microdevices. To extend the utility of COC as an effective microarray substrate, we developed a new method that enabled for the first time in situ synthesis of DNA oligonucleotide microarrays on the COC substrate. To achieve high-quality DNA synthesis, a SiO(2) thin film array was prepatterned on the inert and hydrophobic COC surface using RF sputtering technique. The subsequent in situ DNA synthesis was confined to the surface of the prepatterned hydrophilic SiO(2) thin film features by precision delivery of the phosphoramidite chemistry using an inkjet DNA synthesizer. The in situ SiO(2)-COC DNA microarray demonstrated superior quality and stability in hybridization assays and thermal cycling reactions. Furthermore, we demonstrate that pools of high-quality mixed-oligos could be cleaved off the SiO(2)-COC microarrays and used directly for construction of DNA origami nanostructures. It is believed that this method will not only enable synthesis of high-quality and low-cost COC DNA microarrays but also provide a basis for further development of integrated microfluidics microarrays for a broad range of bioanalytical and biofabrication applications.

Advancing high-throughput gene synthesis technology.

The emerging field of synthetic biology is generating insatiable demands for synthetic genes, which far exceed existing gene synthesis capabilities. This review discusses the current methods of chemical DNA synthesis and gene assembly, as well as the latest engineering tools, technologies and trends which could potentially lead to breakthroughs in the development of accurate, low-cost and high-throughput gene synthesis technology. The capability of generating unlimited supplies of DNA molecules of any sequence or size will transform biomedical research in the near future.