Leader: George Church

The goal of this thrust is to develop a limited number of chassis that should serve a wide range of activities (testbeds). More specifically, we are working toward the following goals:

• determine the components of a chassis necessary to be removed or altered to support our testbeds and devices;
• develop robust (industrial) chassis based on Escherichia coli;
• develop a second chromosome for E.coli that will allow one to integrate large sequences of DNA into the cell and isolate their function from the cell’s native chromosome;
• develop a series of safety control mechanisms on the chassis; and
• develop analytical methods to assay for chassis function and high efficiency and fidelity functioning of devices within the chassis.

Just as in developing the computer it was important to integrate all of the devices (motherboard, disk drive, input-output devices) into a chassis, it is important in the development of synthetic biology to devise a cellular chassis into which we can add the devices constructed in our Devices and Device Composition Thrust and integrate signals from each of these subsystems to enable complex cellular function. The cellular chassis must supply all of the components necessary for cell growth and device function; it must have standard connections so that devices generated to a particular standard can be readily integrated into it; and must be robust enough that it can and will be used by our industrial partners and the broader synthetic biology community for various engineering projects.

Escherichia coli has the advantage that it has an extremely well developed genetic system, it is robust under a variety of growth conditions and can be cultivated on minimal media, its physiology is extremely well studied so that any deviations in physiology that result from the introduction of an engineered device can be easily detected, and various measurement techniques (transcript, protein, metabolite, and metabolic flux profiling as well as many others) have been developed allowing one to pinpoint changes in physiology due to the introduced device. These features will help synthetic biologists predict the likely results when cells are loaded with real circuitry.

Current methods to create cellular chasses require labor-intensive and time-consuming genetic engineering techniques. Moreover, these techniques only permit serial introduction of a single DNA construct into cells at low efficiencies. High-throughput and automated methodologies to rapidly and efficiently make both site-specific and large-scale manipulations of genomes do not currently exist. To address this challenge, we are developing new methods that combine large-scale DNA synthesis techniques with engineered recombination strategies to develop fast and cost–effective construction of de novo gene systems, or cellular chasses. To achieve these cellular chasses goals, we employ a multi-faceted approach:

• genome engineering technology development
• automated and scalable methods of genome engineering (MAGE: Multiplex Automation of Genome Engineering with single-stranded oligonucleotides)
• proof-of-concept biological experiments that leverage the unique expertise of the entire SynBERC team