Team:USP-Brazil/Results/Overview
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Results
To test our toolkit design, we decided to target malaria, a disease of global importance that kills hundreds of thousands by the year. The first step was validating our chosen chassis. We obtained and confirmed that we had Pantoea agglomerans in hands, then proceeded to validate transformation and compatibility with an E. coli origin of replication, to finally try to colonize mosquitoes with it. More details about P. agglomerans identification and transformation can be seen in our Chassis results page. Info about the mosquito colonization experiment is on our Proof of Concept page.
To be able to detect malaria, we designed and tested two systems: one based on the detection of change of the microbiome's RNA expression, using toehold switches, and another sensing the altered metabolites (iron and lactate) in the blood of malaria victims. We were able to validate each of our systems using GFP as a proof of concept, and our experience can be seen here
As we were doing our project focusing on malaria, we based ourselves in previous work in paratransgenesis. We chose and cloned some promising anti-pathogen proteins, like Scorpine and (EPIP)4, {along with our kill switch module’s parts, and submitted them to the parts registry. With Scorpine, we also tested expression and secretion with a pBad/AraC regulated promoter and type 1 secretion system.
To check the feasibility of our design, along with talking to experts, we made a model of the detection and effector modules, trying to simulate time and level of expression of the circuit. See more about modeling here.
But overall, we had a great summer winter, learned a lot from the project and acomplished a lot! Here is a summary of our best experimental achievements:
- Modeling: We modeled the detector triggered production and secretion of effectors.
- Chassis: We characterized our chassis, which is new in iGEM and established transformation protocols.
- Biocontainment: We included our kill switch into the iGEM collection of biobricks.
- Detection: We managed to detect lactate concentrations with our detection system.
- Effector: We successfully produced our effector scorpine and sent various other effector parts to the iGEM collection.
- Mosquito colonization: We were able to see our bacterial chassis glowing in mosquito guts.
