Team:INSA-Lyon/Project/Future direction

From 2010.igem.org

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<h3><font color="purple">Uses</font></h3>
<h3><font color="purple">Uses</font></h3>
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<p>We added a very interesting part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K342002">(phasin-phasin-intein)</a> to the registry which should bind to the PHB granule surface and enable an easier purification way for molecules of interest thanks to the self cleaving protein. Our first goal was to use a Phasin-Phasin-Intein-GFP fusion to test the targeting of that kind of construction  to the granule. But there are also a lot of work to adapt this purification way for proteins or molecule of interest can be carried out further.<br>
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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 <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K342002">(phasin-phasin-intein)</a> wich has 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.  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).
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Concerning the use of the granule as lipid storage, we didn't manage to co-localize the granules and the lycopene inside the bacteria. We couldn't manage to differentiate the lycopene and PHB by fluorescence microscopy. A granules extraction experiment could be perform after having induce lycopene production and then look for lycopene inside those extractions. So this idea has still to be deepen down.
Concerning the use of the granule as lipid storage, we didn't manage to co-localize the granules and the lycopene inside the bacteria. We couldn't manage to differentiate the lycopene and PHB by fluorescence microscopy. A granules extraction experiment could be perform after having induce lycopene production and then look for lycopene inside those extractions. So this idea has still to be deepen down.
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Revision as of 23:48, 27 October 2010





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 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. 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).
Concerning the use of the granule as lipid storage, we didn't manage to co-localize the granules and the lycopene inside the bacteria. We couldn't manage to differentiate the lycopene and PHB by fluorescence microscopy. A granules extraction experiment could be perform after having induce lycopene production and then look for lycopene inside those extractions. So this idea has still to be deepen down.




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.