The various parts, devices and chasses constructed from well-characterized standard biological parts can be integrated to form systems. A system, when comprised within a chassis, will then form an organism capable of performing specified functions. Testbeds, through their need for parts and devices integrated into a chassis, will help drive development of the thrusts. SynBERC currently has three testbeds:

• Tumor-Destroying Bacterium
• Microbial Chemical Factory (including Biofuels)
• Advanced Fermentation Organism

We are dedicated to creating a new field of synthetic biology and helping to build an industry around this area. Thus, we believe that the parts, devices, and chassis used to create the testbeds will form the basis for the field of synthetic biology, and will be extremely important for many future testbeds in SynBERC and to the future synthetic biology industry.

SynBERC testbeds represent "grand challenges" in the design and construction of complex engineered biological systems. Testbeds are selected based on their interest to our industry partners, and the potential for being carried forward by one or more of them. The testbeds represent complex systems that can evaluate the concept of creating independent parts, devices, and chassis created in SynBERC’s thrusts that can be integrated into a functioning whole. Current and future testbeds are chosen to push part and device design beyond the current focus on regulation of gene expression that has characterized most synthetic biology research.

1. Tumor-destroying bacterium (TDB)
Leader: Chris Anderson

The goal of the Tumor-Destroying Bacterium (TDB) testbed is to construct bacteria derived from a non-pathogenic chassis that can be safely injected into the bloodstream, localize to, sense, and invade cancer cells within solid tumors, and deliver a cytotoxic agent to destroy them.  Once constructed, the TDB system will be examined in a B16 melanoma mouse model for evidence of tumor regression.  There is a long history of using either natural bacterial isolates with an intrinsic ability to localize to solid tumors, with immunostimulatory properties, or as live vaccines to treat cancer. Using the principles of ground-up modular design, we take a new approach to constructing these agents.  The products of the TDB testbed will be agents designed to mitigate the limitations of current strategies, but more significantly, will comprise a collection of parts, devices, and chassis that can be reused for other applications.  For example, a device that conveys the ability to deliver a toxic protein to a cancer cell can be reused to construct systems that deliver developmental signals to a stem cell or produce a high-value biopolymer such as silk.  Similarly, devices to direct chemotaxis for penetration of a solid tumor could be reused to direct bioremediation agents to an environmental pollutant.  This testbed is designed to challenge chassis, device, and part design and expose the foundational challenges needed to construct organisms able to sense their environment, integrate signals with well-controlled temporal and logical outputs, and operate within a complex environment.   Moreover, the TDB represents  a grand challenge because the system perform in the most unforgiving environment (the body) and must operate within a critical safety margin.

Current work within the TDB testbed is focused on constructing a first-generation system with the minimal set of genetic devices needed to deliver a protein to B16 melanoma cells within a live mouse. In addition to the hypoxia or cell density-dependent invasion device, this will require 1) a bacterial chassis that is not cleared immediately by the mouse immune system; and 2) a device for delivering a cytotoxic agent after invasion.

2. Microbial chemical factory (MCF)
Leader: Kristala Jones Prather

The goal of this testbed is to construct bacteria with the precursor pathways to enable synthesis of a large variety of natural and unnatural products from renewable resources.  A related objective was to address the lack of standards for incorporation of enzymes and metabolic pathways into the Registry of Standard Biological Parts. While continuing to focus on the development of specific microbial chemical factories, the MCF testbed can also be thought of as a collection of “mini-testbeds” that are explicitly intended to test the function and application of parts and devices evolving from those thrusts.

The objectives of designing organisms that use simple, renewable feedstocks to efficiently produce molecules currently available only by extraction from scarce natural resources or chemicals produced primarily from petroleum feedstocks collectively represent a “grand challenge.” However, we also recognize that meeting this challenge successfully will involve the incorporation of the various tools developed within the Center—which must first be tested and characterized. Small molecule (chemical) production in engineered microbes has a long and rich history, enabling such systems to be used effectively for this purpose. Since the specific molecules chosen for synthesis under the MCF testbed are lacking either known or fully elucidated natural pathways, other biosynthetic pathways are in some instances necessary to provide the appropriate system for functional characterization.

Among the many requirements for development of highly productive microbial chemical factories are the following:

• a naïve host that will not be affected by, or inadvertently transform any intermediates in the biosynthetic pathways into undesirable side products;
• chemical devices (pathways) that will enable synthesis of the basic precursors necessary to synthesize complicated molecules and that will allow the host to consume a variety of inexpensive, renewable carbon sources;
• transporters that will export the molecules out of the cell to reduce their toxicity and make easier their purification; and
• intracellular sensors and regulators that control the flow of metabolites through the chemical synthesis pathways to prevent the accumulation of intermediates or bottlenecks in the biosynthetic pathways.  

Ongoing projects in the MCF testbed are focused on both the production of the target, value-added compounds using Parts and Devices developed through the “top-down” approach, as well as Parts and Devices developed independently within the Thrusts through the “bottom-up” approach. They also include projects that utilize model compounds instead of the two primary targets, to facilitate the “bottom-up” design.  Current, SynBERC-funded projects include improving synthesis of glucaric acid, using enzyme surface display technology to address issues with functional composability in biological synthesis, and the development of a computational design tool to regulate enzyme expression as a means of balancing metabolic flux.

3. Advanced Fermentation Organism
Leader: Christopher Voigt

The goal of this testbed is to apply research from the SynBERC thrusts (parts, devices, chassis) to the construction of a “smart” strain that can be programmed to sense and respond to conditions encountered during a fermentation. The focus will be on the construction of a generic system that is applicable to many potential pathways. E. coli has been chosen as the model system because of the availability of platform parts/devices and genome replacement tools. A goal of the Industrial Testbed is to facilitate interactions between SynBERC’s industrial partners and to organize the research performed in the thrusts around industrial problems. The research plan has been developed from the start in consultation with our industrial partners.

We envision the development and characterization of sensors that can respond to different environmental conditions and common stresses encountered in a fermenter. This will involve both the construction of new sensors as well as the characterization of existing ones such that they can be integrated into a larger genetic program. Genetic circuits will be constructed that enable the integration of potentially dozens of these sensors as inputs and control metabolic pathways and stress responses as outputs. Timers and adaptive circuits will enable the control over the expression of multiple genes at different times during fermentation. A chassis will be developed that is safe, phage resistant, and contains an optimized central metabolism. Mathematical methods will be developed to enable a user to more rapidly insert functional pathways into this organism.

4. Mammalian Testbed: Engineered Stem Cells for Tissues by Design
Leader: Ron Weiss

The ability to regard mammalian cells as programmable entities will result in paradigm shifts for many biomedical applications.  Towards this goal, we propose to establish a SynBERC mammalian testbed that focuses on gaining sophisticated spatiotemporal control over stem cell differentiation for programmed tissue regeneration and maintenance. Current approaches to controlling stem cell differentiation usually focus on careful administration of small molecules (e.g. growth factors), over-expression of one or two cell fate regulators, and embedding molecular cues in customized 3D scaffolds.  While some success has been reported, a variety of inherent limitations appear to prevent these approaches from achieving fully customizable fine-grain spatiotemporal control of cell differentiation. Here we seek to overcome the limitations of existing approaches by incorporating the powerful abstractions and mechanisms of synthetic biology, ultimately allowing us to achieve tissues-by-design.  The emphasis of this testbed is to develop a select set of demonstration systems where stem cells are genetically programmed to differentiate into particular cell types, sometimes in pre-defined spatial configurations, and maintain desired population levels.  At the same time, the development of these systems will motivate important foundational advances in mammalian synthetic biology that will benefit myriad other applications.  The testbed includes relevant mammalian synthetic biology projects in areas that include, but are not limited to, mammalian chassis, parts, devices, and practices.