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Up until now we succeded
• Producing Nanoparticles and loading them
• Clone a pH sensitive promoter
• Engineer a fusion protein between ice nucleation protein and streptavidin

But we didn't have enough time to characterize completely and assemble the parts to form the Taxi.Coli.

What we would have done with more time:

Nanoparticles

• Digestion assay first with commercial MMP2/trypsin then measurement wiht fluorescent release assay using a plate reader (was tested, but we didn't received an important primer in time.
• Eventually try to load the nanoparticles with an actual drug

Cell surface display of Streptavidin

• Cloned a plasmid that encodes a fusion protein INP-Streptavidin-YFP and one INP-YFP-Streptavidin. Then we would have been able to use the exact same protocol we used to characterize the Biobrick BBa_K523013, to proof surface localization.
• Cloned a plasmid that encodes a fusion protein of INP with for example protein A to check if the
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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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