In our project, we chose to work with Pantoea agglomerans as our chassis, and fortunately it has proven to be a very nice chassis! Not only that, we expressed for the first time a plasmid from the pSB1C3 series inside an insect, Aedes aegypti, the Dengue and Zika vector!
Pantoea agglomerans has been used before for paratransgenesis with great success, and we decided to use it. We got a strain isolated from Eucalyptus in our state, which showed good growth and was genetically amenable.
Gram staining of our Pantoea agglomerans. Pretty!
Our first idea was to use Team EPFL 2010 chassis, Asaia sp, as our proof-of-concept chassis. We contacted their P.I. and searched for it in Brazilian laboratories. However, after studying the bureaucracy and thinking carefully about introducing this new species in Brazil, we rethought our approach.
Antibiogram of P. agglomerans. No resistance!! (...from the set of antibiotics that is commonly tested in Enterobacteriaceae)
16S sequencing of our P. agglomerans. Everything checks out!
From left to right: samples of Pantoea sp. X7C, Pantoea sp. X7A and Pantoea agglomerans 33.1 that we recied from our friends over at ESALQ
P. agglomerans growth curve.
We managed to:
- Cultivate and apply protocols for chemical and electrical transformations
- Transform it with pSB1C3 series plasmids
- Express RFP in vitro
- Feed Aedes aegypti with the bacteria and identify expression inside the host vector in vivo!!
These results highlight how feasible (and safe ) it can be for a iGEM team to start a paratransgenesis project completely from scratch! Other chassis can even be more interesting depending on the specifics of what the team is targeting.
In september of this year, a Serratia strain was used for paratransgenesis in a paper published in Science from the group that first used P. agglomerans. And it used some of the same effectors we produced on this project! If you want to see more about alternative chassis, check our conceptual framework page!
P. agglomerans expressing RFP with the iGEM PSB1C3 plasmid. This means it can be transformed with anything on the Registry!
- Wang, Sibao, et al. "Fighting malaria with engineered symbiotic bacteria from vector mosquitoes." Proceedings of the National Academy of Sciences 109.31 (2012): 12734-12739. DOI: 10.1073/pnas.1204158109
- Wang, Sibao, et al. "Driving mosquito refractoriness to Plasmodium falciparum with engineered symbiotic bacteria." Science 357.6358 (2017): 1399-1402. DOI: 10.1126/science.aan5478