In addition to the research thrusts and testbeds, SynBERC has developed a core of collaborative, cross-cutting projects that provide foundational tools and technologies needed to advance synthetic biology both internally and externally. Cross-cutting projects currently include the following:
- automated DNA construction
- models and design
- safety and security
- registries and repositories
1. Automated construction
The goal of the automated construction effort is to develop standard protocols for the automated construction of complex devices consisting of multiple biological parts. More specifically, we are working to:
- develop a series of plasmids to be used in the automated assembly of standard genetic parts;
- develop a protocol employing broth media for selection of plasmid-borne transformants; and
- develop protocols for the use of robots to handle multiple samples for DNA restriction and DNA ligation, plating of bacteria, colony picking and plasmid preparation.
UC Berkeley project. One project, led by Chris Anderson, aims to construct bacterial strains, DNA vectors, and automation protocols for assembling genetic cassettes based on standard assembly. Standard assembly protocols begin with a collection of basic parts encoding functional units of DNA such as ribosome binding sites, promoters, and protein coding sequences. These parts are assembled into larger DNAs using simple cut-and-paste based assembly reactions employing restriction enzymes, ligase, and/or recombinases. These protocols have the potential to 1) eliminate the need for sequencing due to the absence of in vitro polymerization and lack of single-stranded intermediates 2) be very low cost, and 3) be conducted within the cytoplasm of live cells. This final potential provides the possibility that the manual manipulations associated with cloning could ultimately be reduced to simple, small volume transfers eliminating the variability and failure rates associated with complicated procedures. Furthermore, the simplicity of the required operations will enable the use of more scalable and consumable-free acoustic-based liquid handling robotics.
MIT project. Another project, led by Randy Rettberg, is working on the optimization of an automated DNA assembly process developed in the lab of Tom Knight at MIT over the last two years. Many challenges arise when scaling a process up to handle hundreds of assembly reactions per week that are not apparent when conducting the process at lower throughput or by hand. The goals of this project are to identify these process bottlenecks and develop workarounds so that the SynBERC community will have a reliable automated DNA construction process at their disposal to speed the construction of systems for the various thrusts and testbeds.
The current process flow involves five steps:
- Amplification of parts to be assembled via culture growth and DNA extraction
- Preparation of input part DNA for assembly by digesting with restriction enzymes
- Assembly of two input parts with a backbone vector via T4 ligase
- Purification of correctly assembled parts via transformation, plating, and selection on appropriate antibiotic
- Return to (1)
We are developing methods to remove incorrect side products following the ligation reaction. Strepadvidin beads bound to biotinylated oligonucleotides that target the correctly assembled product are being evaluated as a means of purification. Improvements in this area will decrease the number of false positives that result from the assembly process thus reducing the number of colonies that need to be evaluated to find a correctly assembled clone. We are also working to optimize the buffers used for each reaction to ensure maximum activity of restriction and ligation enzymes and also to ensure that carry-over of buffers from previous steps do not inadvertently reduce the efficiency of the digestion, ligation, or transformation reactions. Finally, we are optimizing the protocol so that it will be amenable to both liquid-handling robotics or to manual operation via multi-channel pipettes. We hope this will make the process useful for all SynBERC labs with or without access to liquid-handling robotics.
2. Modeling and design
SynBERC has several modeling and design efforts ongoing at various scales (parts, devices, chassis, metabolic pathways). There are several implicit relationships among those projects. For example, the use of the ReBIT database for the choosing of enzymes to develop novel, synthetic biochemical pathways (Prather), the use of modeling and simulation in the design of ribosome binding (RBS) sites for optimization of gene expression (Voigt), and the use of the ROSETTA protein-modeling program in the design of protein sequences to obtain novel binding and enzymatic properties (Kortemme). ROSETTA uses basic parts (gene [protein] sequences, whereas the RBS calculator/simulator is used at the device level. We are also starting to develop a BioCAD initiative based on an integrated design platform called Clotho, a Java-based toolset designed in a platform-based paradigm to integrate all the computational tools used in synthetic biology research. We will develop the core of Clotho, and integrate into Clotho the design tools for protein and RNA part engineering and the composition tools for predicting protein expression levels. The current applications being employed (e.g. ROSETTA, ReBIT, etc.), as well as new applications for data management, including include sequence viewers/editors, parts database managers, assembly algorithm GUIs, and design tools, will be integrated into Clotho.
3. Safety and security
Whereas all the SynBERC research projects currently underway employ DNA or proteins that are not regarded as biological or chemical dangers, two projects have been designed with the goal of creating “safer” chassis to reduce the risk of spread of potentially dangerous components should such projects be undertaken in the future either in SynBERC or within the synthetic biology community. Tom Knight is characterizing the bacterium Mesoplasma florum and has found that the host does not employ the codon UGA as a translation stop codon, as in prokaryotes and eukaryotes. Thus, DNA can be designed for expression in M. florum that includes the UGA sequences. This DNA will not function properly if placed in E. coli, yeasts or other hosts, thereby enabling safer parts or devices designs. Similary, George Church is removing the stop signals from E. coli through codon redesign for the same purpose. In addition, E. coli is being re-designed to remove phage lysogens and other genetic elements capable of spreading DNA to heterologous hosts, as well as introducing auxotrophy to make the host less capable of growing in non-defined environments. These projects will make E. coli a safer chassis to use with respect to containment of the host and spread of its genetic contents. Safer chassis design is co-ordinated with the human practices thrust. The thrust takes a proactive role in assessing the risks to people, animals, plants, and the environment of potentially toxic DNA and organisms, as well as addressing the role of SynBERC in safe practice to regulatory agencies and the public at large.
4. Registries and repositories
The synthetic biology community and SynBERC are bringing engineering techniques and principles to the engineering of biological systems. One principle is the concept of a reusable biological component - a part, a device, or a system. If parts are to be designed and build by one group but used by a larger community, organized catalogs of those components must be created and a system for parts distribution must be developed.
The Registry of Standard Biological Parts is the first such catalog for the synthetic biology community. It contains information about these components: the identifying number, a short description, long and detailed descriptions, sequences and features, categorization, measured parameters, identity of the designer, references, experience in using the component, and more as needed. The Registry maintains, accepts, and delivers this information from a database at MIT via a web-based user interface. The Registry also maintains the user and group database for its user community, and provides a number of tools for dealing with these components.
The DNA Repository holds the physical DNA for the components that have been submitted by the user community and makes periodic distributions and ships out a complete set of components in the spring to the iGEM teams. It also responds to individual requests for components. The greatest challenge the Registry has is the contribution of high-quality well-documented components. Several community programs are being developed to encourage and reward these contributions. At the same time, we are increasing staff at the Registry to improve the quality of the information and DNA that we provide. We are developing a “Part Promotion Process” which will give ratings to parts and we have formed a quality control process that each part undergoes.
From the early days of synthetic biology, centralization of a catalog of parts has always been considered important so that a critical mass of infrastructure, contributions, and quality could be achieved. Because the synthetic biology community comes from biology, chemistry, chemical engineering and computer science backgrounds, a single registry of parts, like a web site, represents the point of view of a select group of researchers and would fall short of the needs of some groups. We have therefore established the idea of a Web of Registries based on common data interchange formats and ontologies. The development of what is called Internet 2 is based on distribution of content across many sources and an integration of this information by the user interface.
Additionally and importantly, to guide the development of the Registy at MIT and the Web of Registries, (including the JBEI Registry) in areas such as usability, features, tools, the parts data presented, etc., we will establish a Registry Steering/Advisory Committee, to be drawn from the registry user community, to meet periodically to identify and prioritize the goals and activities of the registry groups during future years.