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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 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 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 be 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. Additionally we would have inquiered which other proteins could be fused to INP and still be used to bind nanoparticles.

Sensing/Effector

We succesfully cloned a pH sensitive promoter, and proofed its functionality, but with a bit more time we would have characterized it in more detail. For example repeated the plate reader experiments with more different pHs and even with different buffers.
Furthermore we would have transformed another strain of E.coli (MG1655) with the plasmid encoding the XXY promoter. These promoters originate from E.coli (MG1655) so we suppose that some of the cofactors could be missing in the DHalpha strain we used. In parallel we would have restarted the cloning of the XXY promoters with slight variations of sequences, to check for a better inducible system.

The effector part wasn't very succesful, but we would repeat the purification assay of Gelatinase E and MMP2 too make sure that our negative results aren't due to mistakes made by us following the protocol.
Additionally we would have designed new primers to have a fusion protein between the enzymes and GFP which would facilitate to proof its production.
Eventually after a lot of work we would achieve to clone the sensing promoter and the effector enzymes together into one plasmid. Thus having a plasmid that produces the nanoparticle degrading enzyme when triggered.

Taxi.Coli

Once all the subparts work, we would clone one Biobrick in a backbone that contains another antibiotic resistance, so we could cotransform bacteria with the plasmid respondible for sensing/effector and the plasmid that encodes the Inp-streptavidin fusion protein. Then we would bind the nanoparticles to those Taxi.Coli. The last step to complete the proof of principle we wanted to provide, is to characterize extensively these final version of Taxi.Coli smart drug delivery.

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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