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Synthetic Biology Group

"Synthetic Biology" is not currently well defined in the scientific community. It is clear that it heavily draws upon (as well as contributes back to) other disciplines such as Molecular Biology, Microbiology, Cellular Biology, Genetics, Systems Biology, Biochemistry, Enzymology, Metabolic Engineering, Electrical and Electronics Engineering, Computer Science, Numerical Modeling, Control Theory, and so on. But, how Synthetic Biology is distinct from these related disciplines is more contentious.

 

While many of the goals of Synthetic Biology are shared with these other scientific fields (with the possible exceptions of wholly specifying/designing/implementing new organisms from the genetic base-pair on up, and testing the limits of human comprehension of biological systems through the assertion that "What I cannot create, I do not understand" - R. Feynman), the processes utilized by Synthetic Biologists to achieve these objectives (such as standardization, component characterization and re-usability/interchangeability, and computer-aided design) seem to be reliable hallmarks that set the field apart from its brethren. Although these processes bring with them their own ethical, legal, and societal issues (such as the implications of enabling do-it-yourself biology (DIYbio) communities), they are less often highlighted than some of the more publically controversial objectives of Synthetic Biology which have largely been the focus of the Alfred P. Sloan Foundation and Woodrow Wilson Centers' Synthetic Biology Project.

 

Within the Fuels Synthesis Division, the Synthetic Biology Directorate develops and demonstrates experimental wetware processes and informatic software tools that facilitate, accelerate, standardize, and automate the Molecular Biological tasks shared across all four scientific and technologies divisions at JBEI. These tools are currently collaboratively contributing to the endeavors of the Fuels Synthesis Division's Biofuels Pathways, Metabolic Engineering, Biofuels Toxicity and Tolerance, and Host Engineering Directorates, as well as the Deconstruction Division's Enzyme Optimization Group, and the Feedstock Division's Cell Wall Engineering and Systems Biology Groups. Here, we present three software tools under active development (JBEI-ICE Repository Platform, DeviceEditor, and j5). These three web-based tools fit together in an integrated process, as shown in Figure 1.

 

 

Figure 1. Synthetic Biology: an integrated process. Biological computer-aided design (bioCAD) tools (e.g. DeviceEditor) are utilized to select (from a repository such as JBEI-ICE) biological component "parts" (DNA sequence fragments often associated with biological function(s)) to assemble together. DNA assembly design automation tools (e.g. j5), putatively in conjunction with high-throughput liquid handling robotics or other devices (in collaboration with the Technologies Division's High Throughput Sample Preparation and Enzymology Group Directorates), are then used to facilitate the physical and informatic assembly of the selected component parts into a new part. The assembled product is then deposited in the parts repository, completing the process cycle. Not shown is the very important step of characterizing the newly assembled part (or including it in a library from which to screen/select for desired function), and depositing this performance data along with the part itself in the repository.

The JBEI Parts Registry

In some aspects similar to the MIT Registry of Standard Biological Parts, the JBEI -ICE Repository (Figure 2) serves as a database of biological "parts" (including proper (though not necessarily standardized) parts, but also plasmids, microbial strains, and most recently Arabidopsis seeds, in collaboration with the Feedstocks Division) and the information associated with them. The JBEI Registry provides advanced search features (such as BLAST sequence queries and field-specific filters), tracks the intellectual property and funding sources associated with each part, and (through web-services) facilitates access to parts distributed across multiple labs. Currently under development, access to machine-readable parts characterization data (of particular importance to BioCAD tools) will soon be available. In addition to the parts database, the platform also offers a suite of integrated tools that provide enhanced feature annotation and sequence editing capabilities (Vector Editor), as well as sequence validation automation. The JBEI-ICE Repository Platform software is open source and freely available.

 

 

Figure 2. The JBEI-ICE Repository Platform. (Top left) The parts database stores annotated sequences, along with the other properties associated with each part type. Physical samples, sequencing reaction trace files, and additional classes of information (e.g. characterization data, DNA construction protocols) are easily linked to the part entries, keeping all of the relevant information together in one place. (Bottom) VectorEditor provides full-fledged sequence and annotation editing capabilities, along with ORF prediction, restriction enzyme maps, and smart cloning-aware copy/paste features. (Top right) A sequence validation automation tool visually represents the alignments of sequencing reaction trace files onto the part sequence, allowing the user to rapidly identify mismatches.

DeviceEditor: a visual biological CAD canvas

With the emergence of parts repositories (along with their associated characterization data) and automated DNA assembly methods (Figure 1), bioCAD tools are beginning to play an increasingly important role. Although integration with a retro-synthetic metabolic pathway design tool (a derivative of the GLAMM platform within MicrobesOnline, in collaboration with the Technologies Division's Bioinformatics Analysis Directorate) and externally developed genetic regulation circuitry modeling software (such as ClothoCAD, GenoCAD, Tinkercell, SBW, etc.) is anticipated in the future, DeviceEditor (Figure 3) currently serves as a graphical user interface for designing (combinatorial) DNA assemblies and for generating the corresponding input files required for j5 (see below). Biological parts are mapped to iconic representations either via copy/paste from a highlighted sequence region in Vector Editor (Figure 2, bottom), or from portions of local Genbank-format sequence files. The spatial arrangement of these icons determines the design of the (combinatorial) DNA construct(s) to be assembled. DeviceEditor provides access to controlling the assembly strategy, Eugene design specification rules and the j5-specific parameters used when designing the assembly strategy. It is possible to run j5 directly from within DeviceEditor itself, without necessitating a separate visit to the j5 web interface. DeviceEditor integrates with VectorEditor to graphically display j5-designed assemblies strategies, enabling an iterative and interactive DNA assembly design process.

 

 

Figure 3. DeviceEditor. Serving as a bioCAD canvas, DeviceEditor provides a means to an in silico representation of a biological design schematic (such as that shown above). Each biological part in the design is mapped to a graphic icon (selected from the palette shown at left, which utilizes standardized Synthetic Biology Open Language Visual extension (SBOLv) symbols). Parts are arranged from left to right in the order of assembly from 5' to 3', and combinatorial designs (as shown) are achieved by placing more than one part in the same vertical bin. The panel shown at right provides additional control over the design (e.g. part names and definitions) as well as the DNA assembly strategy (e.g. embedding a part within a primer, or specifying a Golden Gate overhang sequence).

j5: scar-less multi-part DNA assembly design automation

Some of the most important developments within the Synthetic Biology field over the last several years concern vastly improved DNA construction methods. In contrast with traditional multiple cloning site approaches (which are still predominant in practice although the underlying technology has not changed significantly in decades), many new methods have emerged that, through standardization, have greatly enhanced our ability to re-use previous DNA cloning efforts, to productively share constructs with researchers across institutional boundaries, and (perhaps most importantly) to automate the DNA assembly process. In addition to Gateway cloning, and BioBrick assembly (currently very popular in the Synthetic Biology community, especially amongst competitors in the annual iGEM competition), complementary standardized, largely sequence-independent, approaches (such as SLIC/Gibson/CPEC and Golden Gate assembly, described in the j5 user's manual) have been developed that allow for scar-less multi-part DNA construction. While these methods are extremely versatile and powerful, they can require a tedious, error-prone, and labor-intensive design process. In addition, as the price of direct DNA synthesis services decreases, it will be increasingly likely that (perhaps with the exception of combinatorial assemblies) direct synthesis will be more cost-effective than any of these DNA construction methods. We have developed the software package j5 that automates the design of these DNA assembly protocols, determining the most cost-effective assembly strategy (e.g. direct synthesis vs. PCR) as it does so. Furthermore, j5 can condense multiple assembly protocols together, allowing multiple independent DNA construction projects to be executed in parallel in the same 96-well format plates. j5 also produces high-throughput liquid handling robotics platform command scripts to further automate the execution of the DNA assembly protocols.

 

 

Figure 4. j5 DNA assembly design automation software. (Left) The j5 web interface. (Right) The online j5 user's manual.

Useful Links

• Public instance of the JBEI-ICE Repository: Links to the open-source software downloads are provided here.
• j5/DeviceEditor online: The home page for j5 and DeviceEditor. Sign up for no-cost academic/non-profit/government accounts (commercial licenses are available through the LBNL Technology Transfer Department, ttd@lbl.gov). Demonstration movies and links to the user's manuals for j5 and DeviceEditor are also provided here.
• This web site was developed in collaboration with the Technologies Division's Informatics Directorate.