Conclusion and 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.
The first step of our project was to be able to produce Poly-Hydroxy-Butyrate granules.
FIRST STEP : VALIDATED.
We managed to produce granules from a plasmidic operon under natural promoter control. The functional phaC gene of this operon has been sent as a part. We planned to send the complete operon (phaCAB) which would allow any team to produce PHB granules inside E.coli but we could not for technical issues.
In eukaryotic organisms the enzymes required for lipids, like PHB, synthesis are gathered in a unique protein. We intended to find a way to make, as evolution did, a prokaryotic multifonctional enzyme. We performed the first step of this work : comparison of the gene sequences of those enzymes from various species. We saw that a global organisation was conserved. Design and synthesis of such a gene would be the next step of this work.
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 have to be done to determine if the granule purification system presents 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. We found in the registry a part responsible for lycopen synthesis and we decided to test their becoming in a cell producing granules.
Unfortunately, we didn't manage to co-localize the granules and the lycopene inside the bacteria because it was impossible to differentiate the lycopene and PHB by fluorescence microscopy. So, we thought about other ways to verify our hypothesis. For example, granules could have been extracted after induction of lycopene production and analyzed by HPLC. Moreover, a study about the length and the chemical properties of the acyl chain that can be inserted into the granules has to be done to know which kind of lipids can be stocked this way.
The production of our granules was supposed to be under control of curli promoter. We characterized 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 measures to conclude about the performance of our biobrick. However during this characterization, we realized that ompR234 mutation generates a slight increase in the capacity of E. coli to produce biofilm. One interesting thing to do would be to determine if such an effect could be observed with a plasmidic version of this mutation. Indeed, producing this mutated regulator from a high copy plasmid has great chance to amplify the effect observed with the genomic mutation.
Our project was based on our capacity to produce sequentially granules and molecules to insert in those granules. Our system was based on the use of the curli promoter. During the study of this promoter, we realized that a lot of work had to be done to characterize this system. We thought that it was a good opportunity to provide new creative parts to the synthetic biology community. Thus we started this work concurrently to the granule study. We realized too late that it was a bit too ambitious. Our first results make us believe in its great potential even if a lot of work still has to be done.