CRISPRi Transcriptional Gates for Programmable Control of Bacillus subtilis
Presenter: Arya Manda
Faculty Sponsor: Lauren Andrews
School: UMass Amherst
Research Area: Chemical and Biomolecular Engineering
Session: Poster Session 6, 4:15 PM - 5:00 PM, Auditorium, A20
ABSTRACT
Synthetic genetic circuits allow for the precise engineering of dynamic cellular functions and programmable responses using transcriptional regulatory networks. Predictive genetic circuit design using transcriptional logic circuits has been developed in some bacteria, such as Escherichia coli, but has not been established for the industrially relevant Gram-positive bacterium Bacillus subtilis. Our objective is to establish predictive genetic circuit design for B. subtilis. Towards this objective, we designed, constructed, and characterized CRISPRi-based transcriptional NOT gates for B. subtilis, which can be used as building blocks for more complex logic circuits. Each gate output promoter was computationally designed to contain a synthetic operator sequence. The transcriptional gates need to be connected together for complex genetic circuits without cross-reactivity. Therefore, we tested the orthogonality of a subset of five CRISPRi gates and assayed all 25 possible gRNA-promoter pairs. The CRISPRi gates exhibited high specificity and highly repressed only their cognate output promoter. To test composability of the gates, we then used signal matching algorithms to design and model genetic circuits for different logic functions. We built and assayed 15 genetic circuits, including those for NOR, two-input AND, and three-input AND logic functions. The experimental results correlated strongly with the predicted outputs. This validated our approach and use of signal matching algorithm for circuit output predictions in B. subtilis. In future studies, these CRISPRi gates should allow for precise transcriptional control and have wide ranging applications, including therapeutics and bioremediation.