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Up until now we succeded to produce nanoparticles and loading them, to clone a pH sensitive protomer 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 month more

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 Matrixmetalloprotease 2 (MMP2) we would have performed a fluorescent release assay using a plate reader. First we would have used commercial enzymes, to try if the assay works and maybe adjust the protocol, later with the enzymes produced by our own Taxi.Coli.
Eventually we would have also tried to load the nanoparticles with an actual drug.

Cell surface display of Streptavidin

We weren't able to proof 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 have been able to follow the same immunofluorescent staining protocol we succesfully used to characterized the Biobrick BBa_K523013. We actually started this cloning strategy, but didn't have enough time to optimize the PCRs. If this assay proofed surface localization we could conclude that the streptavidin didn't fold correctly or didn't form the tetramers. This would lead us to the inquiery which other protiens could be used to bind the nanoparticles, as for example the protein A.

Sensing/Effector

• Make more plate reader experiments, with more different pHs and differnt Buffers
• Transform other E.coli K12 (MG1655), because the promoter originate from this strain, so maybe the needed xxxy aren't present in the DHalpha strain we use at the moment
• Start new cloning, with slight variations of the sequence
• Redo the purification of the Gelatinase E and of Matrixmetalloprotease2 (MMP2) • Repeat the Cloning strategy with new primers, because those promoters added a stop codon directly after the enzyme, and before the GFP, so to repeat the cloning, would make the visualization much more easier.
• Clone the sensing promoter together with the enzyme to make the plasmid that induces enzyme production upon a signal.

Taxi.Coli

• Cloned one of our Biobricks in a backbone that contains another resistance than chloramphenicol to cotransfect bacteria with the Plasmids responsible for Sensing/Effector and the Plasmid used to connect the Nanoparticles.
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