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Team:INSA-Lyon/Project/Future direction
From 2010.igem.org
Further Directions
Our project was full of ideas but time was short to achieve it completely. So there is still many possibilities and things to do in order to improve our results.
Production
We managed to produce granules and a new operational PhaC part but we didn't send the complete operon (phaCAB) which would able any team to produce PHB granules inside E.coli.
Uses
Granules are a new way to purify proteins and lipids of interest. Our main goal during this project was to develop such a technic. In this context we designed a new part (phasin-phasin-intein) wich has previously been described as a good way to adress protein to the granules. Indeed a protein fused to this part is supposed to be adressed to the granule and once the granules extracted, a switch of pH can then release the protein of interest. Therefore we wanted first to prove that a GFP fused to the phasin-phasin-intein was correctly adressed to the granules. But, due to a lack of time we didn’t manage to realize the experiment. So the first thing to do is to achieve this experiment.
More simple technics already exist to purify proteins and some works has to be done to determine if the granule purification system present any advantage (purity and yield). Simple experiments using GFP as a reporter can be done to complete these studies.
A big challenge in the production of lipids is to find an intracellular vector to stock these highly insoluble molecules. Our hypothesis was that granules with their lipidic composition could be the solution. The first step f interest Granules are mostly made of lipids. Thus our hypothesis was that if we produce lipids in the same cell as granules those lipids would be stocked into the granules.Concerning the use of the granules as lipid storage, we didn't manage to co-localize the granules and the lycopene inside the bacteria. We couldn't differentiate the lycopene and PHB by fluorescence microscopy. Another way to test our hypothesis could have been to extract the granules after induction of lycopene production and analyse them by HPLC.
Regulation
We characterize the natural curli promoter inside a plasmid and we wanted to link those results with the ones of our designed curli promoter. But we were not able to realize the measurements to conclude about the performance of our biobrick.
