Proof of concept
The original aim of the group was to produce bioplastic, P(LA-co-3HB), in our co-culture. Unfortunately, this was not achieved, due to difficulties in integrating the four genes required into B.subtilis.
Despite this, it is still possible to prove the general principle of the bioproduction platform: that S.elongatus can photosynthesise sucrose under inducible conditions and maintain a population of B.subtilis. Therefore we investigated:
- sucrose production of S.elongatus;
- growth of B.subtilis in spent media;
- successful separation of the species in a co-culture container.
More detailed protocols and results can be found on the relevant pages.
Sucrose production of cyanobacteria:
S.elongatus was grown under multiple conditions, with varying concentrations of NaCl, IPTG and chloramphenicol, to ascertain the optimum sucrose production environment. These cultures were grown for four days and aliquots removed and prepared at periodic intervals. The cells were centrifuged and removed in order to quantify only the sucrose that had been exported into the media. The sucrose concentration of these aliquots was analysed using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).
Results were encouraging, and demonstrated that S.elongatus can generously produce and export sucrose under the conditions we hypothesised.
Growth of B.subtilis in spent media:
Since cartridges of B.subtilis must survive in media that has already been used by S.elongatus, we grew cyanobacteria cultures for several days and then removed the cells. This media was then inoculated with B.subtilis and its growth measured using a spectrophotometer. The optical density (OD) suggested that growth was successful, and that B.subtilis will be able to survive in a media-exchange with cyanobacteria like our co-culture.
Separation of the species, growth in co-culture:
Shared media is required by our model, but we wish to separate the two species used for easy module exchange. Most importantly, the B.subtilis had to be kept in its cartridge (S.elongatus in the cartridge will likely not interfere with the extraction or storage processes; B.subtilis in the open media will continue to produce its specific product even after cartridge removal and consume valuable carbon). The sterilised dialysis tubing used was clipped to avoid leaks and filled with B.subtilis, and inserted into the conical flask with media. As hoped, the OD of the cartridge was high as B.subtilis grew quickly, while there was no visible growth in the main media, suggesting that separation was successful.
There was not enough time to prove our concept with a viable product; however, the species are able to exist in a co-culture environment and produce the needed carbon source. This is an excellent base to eventually use in real-world conditions, both terrestrial and otherwise.
We intend to continue the project and fully expect the B.subtilis to produce P(LA-co-3HB) as desired, and eventually other products.