Header

Up until now, we succeeded to produce nanoparticles and to load them, to clone a pH sensitive promoter and to engineer a fusion protein between ice nucleation protein (INP) and streptavidin.
But we neither had the time to characterize our parts extensively nor to assemble the parts to finalize the Taxi.Coli.

What we would have done with 1 more month

Nanoparticles

This part of our project is rather advanced, but there are still some assays that we would have loved to make. For example, to test if and how fast the nanoparticles are digested by GelatinaseE and Matrix metalloprotease 2 (MMP2), we would have performed a fluorescence release assay using a plate reader. First, we would have used commercial enzymes to check that the assay works and maybe adjust the protocol, later with the enzymes produced by the E.coli transformed with the effector plasmid.
Eventually, we would have also tried to load the nanoparticles with an actual drug.

Cell surface display of Streptavidin

We weren't able to prove that the fusion protein between Inp and Streptavidin was exported to the outer membrane. The next step would be to clone a plasmid that encodes a fusion protein between Inp, streptavidin and YFP. Then, we would be able to follow the same immunofluorescent staining protocol we successfully used to characterize the Biobrick BBa_K523013. We actually started this cloning strategy, but didn't have enough time to optimize the PCRs.

We actually used the time between the Jamboree in Lyon and the one at MIT, to clone the two plasmids, and did microscopy using an anti YFP antibody. Check out Cell surface display for further information. The results were promising, but we would need to sequence verify our constructs and repeat the assay several times, improve the antibody concentration and incubation time, for better images. Additionally, we would have inquired which other proteins could be fused to INP and still be used to bind nanoparticles.

Sensing/Effector

We successfully cloned a pH sensitive promoter, and proved its functionality, but, with a bit more time, we would have characterized it in more details and would have been able to find its optimal expression conditions. For example, we would have repeated the plate reader experiments with a wider range of pH values and even with different buffers.
Furthermore, we would have transformed another strain of E.coli (MG1655) from which the promoters (hya and cad) originate with the plasmid. This would assure that the necessary transcription factors and activators are present and can initiate transcription of the superfolded GFP. In parallel, we would have restarted the cloning of the hya and cad promoters with slight variations of sequences, to check for a better inducible system.

The effector part wasn't very successful, but we would repeat the purification assay of Gelatinase E and MMP2 to make sure that our negative results aren't due to manipulations error.
Additionally, we would have designed new primers to have a fusion protein between the enzymes and GFP which would facilitate to prove its production.

Eventually, we would clone the sensing promoter and the effector enzymes together into one plasmid. Thus having a plasmid that produces the nanoparticle degrading enzyme when triggered by a pH change, as in the original idea.

Taxi.Coli

Once all the subparts worked, we would clone one Biobrick made out of our promoter and our gelatinase gene in one backbone. This backbone would then contain a different antibiotic resistance than our Cell-surface display plasmid so that we could co-transform bacteria with the plasmid responsible for sensing/effector and the plasmid that encodes the Inp-streptavidin fusion protein. Then, we would bind the nanoparticles to those Taxi.Coli bacteria.
The last step to complete the proof of principle we wanted to provide is to characterize extensively this final version of Taxi.Coli smart drug delivery.