Synthetic biology can improve paratransgenesis by bringing an engineering approach to this technique, especially modeling and modularity. Our system contains four genetic modules designed for Bacillus subtilis, a chassis that lives inside the midgut of Lutzomyia longipalpis. The Protection, Detection, Elimination and Biocontainment modules were designed considering the physiological specificities of the host, the vector and the chassis, including biosafety aspects, making our strategy as feasible and responsible as possible for a real application.
Chassis
A good chassis makes a good system. We selected B. subtilis to carry our circuit and act against the parasite within the vector. Being a natural commensal organism of L. longipalpis microbiota and Generally Recognized as Safe (GRAS) are reasons that make B. subtilis an ideal choice of chassis.
Protection
Increasing the survival of our chassis in the sugar baits until the ingestion by the vector guarantees an efficient application in the field. Protection module is a circuit used to keep the chassis in its sporulated form in the sugar baits and induce germination only when inside the midgut.
Detection
Specificity is a key factor when working with strategies that involve the spread of engineered bacteria in the environment. The Detection module is responsible for this specificity in our system, defining the conditions for the activation of the anti-leishmania circuit. Utilizing the B. subtilis ability to uptake nucleic acids from the environment, we designed a riboswitch-based circuit from a "de novo" construct of Toehold RNA Switches specifically for miRNAs found in the midgut of L. longipalpis.
Elimination
Fast and Modular. These are the characteristics we looked for in this module. The elimination module is responsible for the production of Antimicrobial Peptides (AMPs), our leishmanicidal molecules, in its inactivated form and for the activation of these peptides when in contact with enzymes produced by the sandfly after a blood meal. This is necessary because the AMPs are toxic for bacterias too, so they have to be activated only when necessary
Biocontainment
The Biocontainment device seeks to prevent the uncontrolled spread of our chassis into the environment. We designed an innovative Kill Switch based on light-inducible CRISPR/Cpf1 and together with the FCB-UANL team, we created a compilation of Potential Cell Lysis Mechanisms for B. subtilis