Biomolecular Breadboards for Prototyping and Debugging Synthetic Biocircuits

This is a joint project between Richard Murray (Caltech), Vincent Noireaux (U. Minnesota) and Paul Rothemund (Caltech), funded by the DARPA Living Foundries program. The information on this page focuses primarily on the work involving my research group. Additional information about this project is available on our OpenWetWare web pages.

Current participants: Zachary Sun (PhD student, Biology)* Clarmyra Hayes (research technician) Jongmin Kim (postdoc, BE)* Dan Siegal (-Gaskins) (postdoc, BE → Schafer Corp)* Anu Thubagere (PhD student, BE)* Gita Mahmoudabadi (PhD student, BE)

Collaborators Paul Rothemund (Caltech) Vincent Noireaux (U. Minnesota)

* Partially supported

Objectives

Overview of the cell-free expression breadboard process.

The goal of this project is to build a set of “biomolecular breadboards” to create a systematic, engineering-oriented approach to synthesizing biomolecular circuits that involves developing, modeling, and debugging a sequence of prototype devices, each at increasing levels of complexity and each allowing the incorporation of increasingly realistic operating environments for either in vitro or in vivo applications. We are adapting an existing cell-free toolbox developed at U. Minnesota to create a set of prototyping environments for testing biological circuits. A sequence of increasingly complex environments, ending in prokaryotic cells, will be used to demonstrate the ability to prototype circuits that function in vivo, with iteration in in vitro assays and models to streamline development of predictable, in vivo functionality.

• Objective 1: Post protocols for producing basic cell-free breadboard on public web site along with summary of costs. Provide sequences for positive controls.
• Objective 2: Demonstrate the use of cell-free breadboard on 2 existing circuits (chosen from negatively autoregulated gene, simple genetic switch, oscillator, feedforward loop) and document time required to implement a simple circuit and perform design iterations.
• Objective 3: Post complete documentation of a transcription-translation cell-free platform that includes a set of transcriptional repression units (non-degradable and degradable versions), the working principles of the toolbox (procedures and protocols), and validated models for the repressors based on in vitro and in vivo characterization.
• Objective 4: Demonstrate the design of a simple circuit (3–6 unique promoters) that consists of a new set of genetic elements, documenting the interactions, conditions, and compensation mechanisms required to obtain a working circuit in E. coli. Investigators shall provide an analysis of the design cycle times compared to iGEM standard assembly protocol and Gibson- based protocol (using in vivo testing and debugging). Target 3 day prototyping cycle time and 1 month from design to implementation, with the ability to test 25 circuit variants simultaneously.

References

• DARPA Living Foundries kickoff presentation, 12 Jul 2012