Fluorescent amplification for next generation sequencing (FA-NGS) library preparation.

BACKGROUND:Next generation sequencing (NGS) has become a universal practice in modern molecular biology. As the throughput of sequencing experiments increases, the preparation of conventional multiplexed libraries becomes more labor intensive. Conventional library preparation typically requires quality control (QC) testing for individual libraries such as amplification success evaluation and quantification, none of which occur until the end of the library preparation process. RESULTS:In this study, we address the need for a more streamlined high-throughput NGS workflow by tethering real-time quantitative PCR (qPCR) to conventional workflows to save time and implement single tube and single reagent QC. We modified two distinct library preparation workflows by replacing PCR and quantification with qPCR using SYBR Green I. qPCR enabled individual library quantification for pooling in a single tube without the need for additional reagents. Additionally, a melting curve analysis was implemented as an intermediate QC test to confirm successful amplification. Sequencing analysis showed comparable percent reads for each indexed library, demonstrating that pooling calculations based on qPCR allow for an even representation of sequencing reads. To aid the modified workflow, a software toolkit was developed and used to generate pooling instructions and analyze qPCR and melting curve data. CONCLUSIONS:We successfully applied fluorescent amplification for next generation sequencing (FA-NGS) library preparation to both plasmids and bacterial genomes. As a result of using qPCR for quantification and proceeding directly to library pooling, the modified library preparation workflow has fewer overall steps. Therefore, we speculate that the FA-NGS workflow has less risk of user error. The melting curve analysis provides the necessary QC test to identify and troubleshoot library failures prior to sequencing. While this study demonstrates the value of FA-NGS for plasmid or gDNA libraries, we speculate that its versatility could lead to successful application across other library types

Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3' Terminal Structures for Enzymatic De Novo DNA Synthesis.

Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3' terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin

DeviceEditor visual biological CAD canvas

<p>Abstract</p> <p>Background</p> <p>Biological Computer Aided Design (bioCAD) assists the <it>de novo </it>design and selection of existing genetic components to achieve a desired biological activity, as part of an integrated design-build-test cycle. To meet the emerging needs of Synthetic Biology, bioCAD tools must address the increasing prevalence of combinatorial library design, design rule specification, and scar-less multi-part DNA assembly.</p> <p>Results</p> <p>We report the development and deployment of web-based bioCAD software, DeviceEditor, which provides a graphical design environment that mimics the intuitive visual whiteboard design process practiced in biological laboratories. The key innovations of DeviceEditor include visual combinatorial library design, direct integration with scar-less multi-part DNA assembly design automation, and a graphical user interface for the creation and modification of design specification rules. We demonstrate how biological designs are rendered on the DeviceEditor canvas, and we present effective visualizations of genetic component ordering and combinatorial variations within complex designs.</p> <p>Conclusions</p> <p>DeviceEditor liberates researchers from DNA base-pair manipulation, and enables users to create successful prototypes using standardized, functional, and visual abstractions. Open and documented software interfaces support further integration of DeviceEditor with other bioCAD tools and software platforms. DeviceEditor saves researcher time and institutional resources through correct-by-construction design, the automation of tedious tasks, design reuse, and the minimization of DNA assembly costs.</p

Adenosine Triphosphate and Carbon Efficient Route to Second Generation Biofuel Isopentanol.

Climate change necessitates the development of CO2 neutral or negative routes to chemicals currently produced from fossil carbon. In this paper we demonstrate a pathway from the renewable resource glucose to next generation biofuel isopentanol by pairing the isovaleryl-CoA biosynthesis pathway from Myxococcus xanthus and a butyryl-CoA reductase from Clostridium acetobutylicum. The best plasmid and Escherichia coli strain combination makes 80.50 ± 8.08 (SD) mg/L of isopentanol after 36 h under microaerobic conditions with an oleyl alcohol overlay. In addition, the system also shows a strong preference for isopentanol production over prenol in microaerobic conditions. Finally, the pathway requires zero adenosine triphosphate and can be paired theoretically with nonoxidative glycolysis, the combination being redox balanced from glucose thus avoiding unnecessary carbon loss as CO2. These pathway properties make the isovaleryl-CoA pathway an attractive isopentanol production route for further optimization

Evidence for a Monomeric Structure of Nonribosomal Peptide Synthetases

AbstractNonribosomal peptide synthetases (NRPS) are multimodular biocatalysts that bacteria and fungi use to assemble many complex peptides with broad biological activities. The same modular enzymatic assembly line principles are found in fatty acid synthases (FAS), polyketide synthases (PKS), and most recently in hybrid NRPS/PKS multienzymes. FAS as well as PKS are known to function as homodimeric enzyme complexes, raising the question of whether NRPS may also act as homodimers. To test this hypothesis, biophysical methods (size exclusion chromatography, analytical equilibrium ultracentrifugation, and chemical crosslinking) and biochemical methods (two-affinity-tag-system and complementation studies with enzymes being inactivated in different catalytic domains) were applied to NRPS subunits from the gramicidin S (GrsA-ATE), tyrocidine (TycB1-CAT and TycB2-3-AT.CATE), and enterobactin (EntF-CATTe) biosynthetic systems. These methods had revealed the dimeric structure of FAS and PKS previously, but all three NRPS systems investigated are functionally active as monomers

Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis

Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 30 terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin

End-to-end automated microfluidic platform for synthetic biology: from design to functional analysis

DNAConstructor scripts.zip. Zip file containing files gfp_DNAConstructor.txt and rfp_DNAConstructor.txt, input script files for DNA Constructor. (ZIP 1.7 kb

A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae

Despite the extensive use of&nbsp;Saccharomyces cerevisiae&nbsp;as a platform for synthetic biology, strain engineering remains slow and laborious. Here, we employ CRISPR/Cas9 technology to build a cloning-free toolkit that addresses commonly encountered obstacles in metabolic engineering, including chromosomal integration locus and promoter selection, as well as protein localization and solubility. The toolkit includes 23 Cas9-sgRNA plasmids, 37 promoters of various strengths and temporal expression profiles, and 10 protein-localization, degradation&nbsp;and solubility tags.&nbsp;We facilitated the use of these parts via a web-based tool, that automates the generation of DNA fragments for integration. Our system builds upon existing gene editing methods in the thoroughness with which the parts are standardized and characterized, the types and number of parts available and the ease with which our methodology can be used to perform genetic edits in yeast. We demonstrated the applicability of this toolkit by optimizing the expression of a challenging but industrially important enzyme, taxadiene synthase (TXS). This approach enabled us to diagnose an issue with TXS solubility, the resolution of which yielded a 25-fold improvement in taxadiene production

Biochemical Characterization of β-Amino Acid Incorporation in Fluvirucin B₂ Biosynthesis

Naturally occurring lactams, such as the polyketide-derived macrolactams, provide a diverse class of natural products that could enhance existing chemically produced lactams. Although β-amino acid loading in the fluvirucin B2 polyketide pathway was proposed by a previously identified putative biosynthetic gene cluster, biochemical characterization of the complete loading enzymes has not been described. Here we elucidate the complete biosynthetic pathway of the β-amino acid loading pathway in fluvirucin B2 biosynthesis. We demonstrate the promiscuity of the loading pathway to utilize a range of amino acids and further illustrate the ability to introduce non-native acyl transferases to selectively transfer β-amino acids onto a polyketide synthase (PKS) loading platform. The results presented here provide a detailed biochemical description of β-amino acid selection and will further aid in future efforts to develop engineered lactam-producing PKS platforms