Team:MIT/Circuit
Project
Circuit
Context
Our system is a cheating co-culture. C. hutchinsonii spends its own resources to break down cellulose into simple sugars, and E. coli, the cheater, consumes the sugars for free. Traditionally, cheaters become predominant in a multi-species community because they have a higher level of competitive fitness.
The Challenge
Our naive co-culture experiment showed that C. hutchinsonii actually dominates the community. We attributed this to C. hutchinsonii’s efficiency in taking up the sugars it produces, since these sugars need to diffuse into solution to become available to E. coli. The additional metabolic burden on E. coli when it has to produce biofuel would only exacerbate the high proportion of C. hutchinsonii, but since we hoped to create an efficient system for consolidated bioprocessing, we wanted more E. coli to generate more product. Our goal was to create a circuit that would stabilize populations with a larger proportion of E. coli in the co-culture.
The Circuit
RelE, a suicide gene, is constitutively expressed in C. hutchinsonii. At high E. coli levels, LuxI is expressed to produce 3OC6HSL, which diffuses in the solution and activates the antidote gene RelB, rescuing C. hutchinsonii from the RelE. Therefore, C. hutchinsonii relies on a certain concentration of E. coli to survive.
Since E. coli’s dependence on C. hutchinsonii creates a natural one-way metabolic link between E. coli and C. hutchinsonii, we designed a circuit to create dependency in the other direction - in other words, we incentivized C. hutchinsonii to allow more E. coli to grow. In our system, C. hutchinsonii and E. coli constitutively express RelE (a suicide gene) and LuxI (produces signal molecule 3OC6HSL) respectively. At high enough E. coli levels, diffused 3OC6HSL activates the antidote gene RelB in C. hutchinsonii, rescuing it from the RelE. Therefore, C. hutchinsonii requires a certain concentration of E. coli in order to survive. To adjust the population ratios, we would tune the strength of the RBS and promoter of RelE, LuxR and LuxI.
Our general circuit design was inspired by examples of published systems, including the synthetic E. coli system by Balagaddé et al. (2008) and the programmed population control system of You et al. (2004). We chose to use the Lux system, originally isolated from vibrio fischeri, since it is one of the most widely used AHL signaling systems. We chose RelE/RelB as our toxin-antitoxin system because other systems are more specific to E. coli and less likely to be effective in C. hutchinsonii. While the Lux and RelE/RelB systems have been demonstrated to function in E. coli, the novelty in our work lies in how we combined them and designed the system for expression in C. hutchinsonii.
Our general circuit design was inspired by examples of published systems, including the synthetic E. coli system by Balagaddé et al. (2008) and the programmed population control system of You et al. (2004). We chose to use the Lux system, originally isolated from vibrio fischeri, since it is one of the most widely used AHL signaling systems. We chose RelE/RelB as our toxin-antitoxin system because other systems are more specific to E. coli and less likely to be effective in C. hutchinsonii. While the Lux and RelE/RelB systems have been demonstrated to function in E. coli, the novelty in our work lies in how we combined them and designed the system for expression in C. hutchinsonii.
