A Procedural Framework for Benchmarking Biofoundry Capabilities

Benchmarking compares the performance of a product or service with a competitor. In a biofoundry context, capability benchmarking enables more effective use of development resources and furthering business development efforts. Biofoundries considering benchmarking activities are immediately faced with many implementation questions and decisions. While differing circumstances between biofoundries may lead to different answers to those same questions, a common framework for the benchmarking process is desirable. Perhaps the framework described here, and developed for the United States Department of Energy Agile BioFoundry, will be useful to other biofoundries around the world

Streamlining the Design-to-Build Transition with Build-Optimization Software Tools

Scaling-up capabilities for the design, build, and test of synthetic biology constructs holds great promise for the development of new applications in fuels, chemical production, or cellular-behavior engineering. Construct design is an essential component in this process; however, not every designed DNA sequence can be readily manufactured, even using state-of-the-art DNA synthesis methods. Current biological computer-aided design and manufacture tools (bioCAD/CAM) do not adequately consider the limitations of DNA synthesis technologies when generating their outputs. Designed sequences that violate DNA synthesis constraints may require substantial sequence redesign or lead to price-premiums and temporal delays, which adversely impact the efficiency of the DNA manufacturing process. We have developed a suite of build-optimization software tools (BOOST) to streamline the design-build transition in synthetic biology engineering workflows. BOOST incorporates knowledge of DNA synthesis success determinants into the design process to output ready-to-build sequences, preempting the need for sequence redesign. The BOOST web application is available at https://boost.jgi.doe.gov and its Application Program Interfaces (API) enable integration into automated, customized DNA design processes. The herein presented results highlight the effectiveness of BOOST in reducing DNA synthesis costs and timelines

PaR-PaR Laboratory Automation Platform

Labor-intensive multistep biological tasks, such as the construction and cloning of DNA molecules, are prime candidates for laboratory automation. Flexible and biology-friendly operation of robotic equipment is key to its successful integration in biological laboratories, and the efforts required to operate a robot must be much smaller than the alternative manual lab work. To achieve these goals, a simple high-level biology-friendly robot programming language is needed. We have developed and experimentally validated such a language: Programming a Robot (PaR-PaR). The syntax and compiler for the language are based on computer science principles and a deep understanding of biological workflows. PaR-PaR allows researchers to use liquid-handling robots effectively, enabling experiments that would not have been considered previously. After minimal training, a biologist can independently write complicated protocols for a robot within an hour. Adoption of PaR-PaR as a standard cross-platform language would enable hand-written or software-generated robotic protocols to be shared across laboratories

Structure of C₂₀ [5]-ladderane fatty acid, and the proposed major steps of the ladderane biosynthetic pathway.

<p>desaturation of acyl-ACPs to form polyunsaturated (all-<i>trans</i>) intermediates and cyclization <i>via</i> a radical cascade mechanism (adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151087#pone.0151087.ref011" target="_blank">11</a>]).</p

Synthetic operons used in this study.

<p>Synthetic operons used in this study.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Growth and fatty acid profiles for strain expressing operons 1 and 2 and control strain.

<p>(A) Growth curve of ladd-initial and control strains. (B) GC/MS total ion chromatograms (TIC) of fatty acids extracted from ladd-initial and control strains post-cultivation and subjected to methyl ester derivatization. The most prominent fatty acid methyl esters are labeled with numbers: 1, C14:1; 2, C14:0; 3, C16:1; 4, C16:0; 5, C17 cyclopropane fatty acid (CFA); 6, C18:1; 7, C18:0; 8, C19 CFA.</p

DNA assembly scheme for construction of operons 3–11 (see Table 2 for additional detail).

<p>Each operon has a unique P_tet promoter, bicistronic design (BCD) element, and terminator chosen from the BIOFAB database. Restriction sites in each final operon plasmid allow for efficient, modular assembly of multiple operons in a final vector, such as a bacterial artificial chromosome or fosmid.</p

<i>In vivo</i> tests of function in putative phytoene desaturases from <i>K</i>. <i>stuttgartiensis</i> (kuste3336 and kuste3607).

<p>(Left) Phytoene desaturation to lycopene catalyzed by CrtI and schematic of the pLyc vector. (Right) Lycopene production in <i>E</i>. <i>coli</i> MG1655 strains (from left to right): Lyc (positive control), Lyc36 (<i>crtI</i> in pLyc replaced with kuste3336), Lyc07 (<i>crtI</i> in pLyc replaced with kuste3607), and Lyc-no-CrtI (negative control with <i>crtI</i> gene removed) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151087#pone.0151087.t001" target="_blank">Table 1</a> for details on strains).</p

A Droplet Microfluidic Platform for Automating Genetic Engineering

We present a water-in-oil droplet microfluidic platform for transformation, culture and expression of recombinant proteins in multiple host organisms including bacteria, yeast and fungi. The platform consists of a hybrid digital microfluidic/channel-based droplet chip with integrated temperature control to allow complete automation and integration of plasmid addition, heat-shock transformation, addition of selection medium, culture, and protein expression. The microfluidic format permitted significant reduction in consumption (100-fold) of expensive reagents such as DNA and enzymes compared to the benchtop method. The chip contains a channel to continuously replenish oil to the culture chamber to provide a fresh supply of oxygen to the cells for long-term (∼5 days) cell culture. The flow channel also replenished oil lost to evaporation and increased the number of droplets that could be processed and cultured. The platform was validated by transforming several plasmids into Escherichia coli including plasmids containing genes for fluorescent proteins GFP, BFP and RFP; plasmids with selectable markers for ampicillin or kanamycin resistance; and a Golden Gate DNA assembly reaction. We also demonstrate the applicability of this platform for transformation in widely used eukaryotic organisms such as <i>Saccharomyces cerevisiae</i> and <i>Aspergillus niger</i>. Duration and temperatures of the microfluidic heat-shock procedures were optimized to yield transformation efficiencies comparable to those obtained by benchtop methods with a throughput up to 6 droplets/min. The proposed platform offers potential for automation of molecular biology experiments significantly reducing cost, time and variability while improving throughput