Eugene – A Domain Specific Language for Specifying and Constraining Synthetic Biological Parts, Devices, and Systems

BACKGROUND: Synthetic biological systems are currently created by an ad-hoc, iterative process of specification, design, and assembly. These systems would greatly benefit from a more formalized and rigorous specification of the desired system components as well as constraints on their composition. Therefore, the creation of robust and efficient design flows and tools is imperative. We present a human readable language (Eugene) that allows for the specification of synthetic biological designs based on biological parts, as well as provides a very expressive constraint system to drive the automatic creation of composite Parts (Devices) from a collection of individual Parts. RESULTS: We illustrate Eugene's capabilities in three different areas: Device specification, design space exploration, and assembly and simulation integration. These results highlight Eugene's ability to create combinatorial design spaces and prune these spaces for simulation or physical assembly. Eugene creates functional designs quickly and cost-effectively. CONCLUSIONS: Eugene is intended for forward engineering of DNA-based devices, and through its data types and execution semantics, reflects the desired abstraction hierarchy in synthetic biology. Eugene provides a powerful constraint system which can be used to drive the creation of new devices at runtime. It accomplishes all of this while being part of a larger tool chain which includes support for design, simulation, and physical device assembly

BBF RFC 94: Type IIS Assembly for Bacterial Transcriptional Units: A Standardized Assembly Method for Building Bacterial Transcriptional Units Using the Type IIS Restriction Enzymes BsaI and BbsI

This RFC94 describes an assembly standard based on the Type IIS restriction enzymes BsaI and BbsI (also called BpiI). This assembly standard is based upon the Modular Cloning (MoClo) assembly strategy, which was introduced in 2011 by Weber et al. [1] and is based upon Golden Gate cloning [2]. In this RFC, we describe our proposed MoClo standard for generating a library of bacterial DNA parts for generating four-part transcriptional units (promoter : 5’UTR : CDS : 3’UTR). In this work, we define 5’UTRs as including ribosomal binding sites (RBS) and bi-cistronic design elements (BCDs) [3], and 3’UTRs as transcriptional terminators. The 2012-2014 BostonU iGEM teams completed this work and a more compact library has also been created based on this work [4]

Synthetic Biology Open Language (SBOL) Version 1.1.0

In this BioBricks Foundation Request for Comments (BBF RFC), we specify the Synthetic Biology Open Language (SBOL) Version 1.1.0 to enable the electronic exchange of information describing DNA components used in synthetic biology. We define: 1. the vocabulary, a set of preferred terms and 2. the core data model, a common computational representation

Buildout and integration of an automated high-throughput CLIA laboratory for SARS-CoV-2 testing on a large urban campus

In 2019, the first cases of SARS-CoV-2 were detected in Wuhan, China, and by early 2020 the first cases were identified in the United States. SARS-CoV-2 infections increased in the US causing many states to implement stay-at-home orders and additional safety precautions to mitigate potential outbreaks. As policies changed throughout the pandemic and restrictions lifted, there was an increase in demand for COVID-19 testing which was costly, difficult to obtain, or had long turn-around times. Some academic institutions, including Boston University (BU), created an on-campus COVID-19 screening protocol as part of a plan for the safe return of students, faculty, and staff to campus with the option for in-person classes. At BU, we put together an automated high-throughput clinical testing laboratory with the capacity to run 45,000 individual tests weekly by Fall of 2020, with a purpose-built clinical testing laboratory, a multiplexed reverse transcription PCR (RT-qPCR) test, robotic instrumentation, and trained staff. There were many challenges including supply chain issues for personal protective equipment and testing materials in addition to equipment that were in high demand. The BU Clinical Testing Laboratory (CTL) was operational at the start of Fall 2020 and performed over 1 million SARS-CoV-2 PCR tests during the 2020-2021 academic year.Boston UniversityPublished versio

Hardware, Software, and Wetware Codesign Environment for Synthetic Biology

Synthetic biology is the process of forward engineering living systems. These systems can be used to produce biobased materials, agriculture, medicine, and energy. One approach to designing these systems is to employ techniques from the design of embedded electronics. These techniques include abstraction, standards, modularity, automated design, and formal semantic models of computation. Together, these elements form the foundation of “biodesign automation,” where software, robotics, and microfluidic devices combine to create exciting biological systems of the future. This paper describes a “hardware, software, wetware” codesign vision where software tools can be made to act as “genetic compilers” that transform high-level specifications into engineered “genetic circuits” (wetware). This is followed by a process where automation equipment, well-defined experimental workflows, and microfluidic devices are explicitly designed to house, execute, and test these circuits (hardware). These systems can be used as either massively parallel experimental platforms or distributed bioremediation and biosensing devices. Next, scheduling and control algorithms (software) manage these systems’ actual execution and data analysis tasks. A distinguishing feature of this approach is how all three of these aspects (hardware, software, and wetware) may be derived from the same basic specification in parallel and generated to fulfill specific cost, performance, and structural requirements

Reducing DNA context dependence in bacterial promoters

<div><p>Variation in the DNA sequence upstream of bacterial promoters is known to affect the expression levels of the products they regulate, sometimes dramatically. While neutral synthetic insulator sequences have been found to buffer promoters from upstream DNA context, there are no established methods for designing effective insulator sequences with predictable effects on expression levels. We address this problem with Degenerate Insulation Screening (DIS), a novel method based on a randomized 36-nucleotide insulator library and a simple, high-throughput, flow-cytometry-based screen that randomly samples from a library of 4³⁶ potential insulated promoters. The results of this screen can then be compared against a reference uninsulated device to select a set of insulated promoters providing a precise level of expression. We verify this method by insulating the constitutive, inducible, and repressible promotors of a four transcriptional-unit inverter (NOT-gate) circuit, finding both that order dependence is largely eliminated by insulation and that circuit performance is also significantly improved, with a 5.8-fold mean improvement in on/off ratio.</p></div

Comparison of GFP and RFP fold changes upon induction with L-arabinose of the 24-inverter permutation with no insulators.

<p>Most circuit permutations fail to provide anything close to the anticipated behavior, and even closely related circuits can yield radically different behavior.</p

Reducing DNA context dependence in bacterial promoters - Fig 1

<p>(a) Degenerate Insulation Screening (DIS) protocol for insulation of promoters. The DIS protocol produces spacers with a wide range of expression behaviors, including many matches to a given reference level and variants that can be used for tuning expression: (b) distribution of GFP expression from samples of 36 nt insulated J23100_E-GFP cassette library, in units of Molecules of Equivalent FLuorescein (MEFL); (c) distribution of fold induction of GFP expression from samples of inducible 36 nt insulated pBAD_F-GFP library upon induction with L-arabinose. (d) distribution of fold repression of RFP expression from samples of repressible 36 nt insulated pTet_A-RFP library upon induction of repressor expression with L-arabinose. Red bar indicates performance identical to reference device; orange bars mark samples with ≥10X induction/repression. Plus and minus distributions for pBAD and pTet are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176013#pone.0176013.s005" target="_blank">S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176013#pone.0176013.s006" target="_blank">S5</a> Figs, respectively.</p

Efficacy of DIS promoter insulation is tested through permutation of a bacterial, monocistronic, transcriptional TetR-“inverter” circuit composed of 4 tandem transcriptional units assembled by means of the MoClo DNA assembly method.

<p>(a) shows the “base” design that is permuted, in which constitutive expression of AraC is induced by L-Arabinose, relieving its repression of the pBAD promoter and resulting in expression of the TetR repressor and GFP. TetR, in turn, represses the pTet promoter, reducing the expression of RFP. Thus, when “on” this circuit should have high green and low red fluorescence, and when “off” it should have low green and high red. Circles between transcriptional units represent the 4 bp scars left by MoClo assembly at the edges of each transcriptional unit in the final construct. (b) A set of 24 permutations are created, covering all possible orderings of transcriptional units, holding orientation constant. As the contents of each transcriptional unit are the same, any differences in behavior are expected to be due to differences in genetic context, and should be able to be eliminated by effective insulators.</p