Team:Nantes/Demonstrate
Proof of concept : a new way of controlling gene expression in time.
We have designed simple insert constructs (each containing 1 sugar specific promoter and 1 fluorescent reporter gene) pLAC, pARA and pRIB that have been characterized in the most common conditions : at room temperature and at a neutral pH (=7,2).
Characterizing the expression of the fluorescent protein associated to a sugar promoter allows us to predict the moment of expression and the length of expression of any gene that could be inserted instead of a fluorescent reporter gene.
From our lab work results, we can confirm that the Arabinose promoter (pARA), that has been associated with the Cyan Fluorescent Protein (CFP), is active around 4.5 hours after the addition of 0.2% arabinose, and this activation lasts around 12.5 hours.
Figure 1: Rate of CFP Fluorescence/hours for E.coli K12 MG1655 transformed with pARA-CFP simple insert in the absence or in the presence of 0,2% of Arabinose
The promoter associated with lactose (pLAC), that has been associated with the Green Fluorescent Protein (CFP), is active around 2.5 hours after the addition of 0.2% lactose, and this activation lasts around 4.5 hours.
Figure 2: Rate of GFP Fluorescence/hours for E.coli K12 MG1655 transformed with pLAC-GFP simple insert in the absence or in the presence of 0,2% of Lactose
The promoter associated with ribose (pRIB), that has been associated with the Yellow Fluorescent Protein (YFP), is active around 3 hours after the addition of 0.2% ribose, and this activation lasts around 7.5 hours.
Figure 3: Rate of YFP Fluorescence/hours for E.coli K12 MG1655 transformed with pRIB-YFP simple insert in the absence or in the presence of 0,2% of Ribose
These results show that our tool is capable of controlling gene expression in time. The backbone of our constructs was created to give to the user the opportunity to insert any gene of interest under the control of these promoters, replacing the fluorescent reporter genes. This tool can be used in any industry, company or lab that needs a specific time of expression for the gene that they want to express. Our tool is cheap, only requiring the user to buy materials such as sugar, furthermore the tool does not require fossil energy to operate #EnvironementFriendly
Improving our tool : a step towards a finer and a more complete regulation of gene expression in time.
To make our project more complete, and to offer the possibility of predicting expression under different conditions (temperature, pH, sugar concentration in the medium) and in a hierarchical way, we worked on the design of a plasmid containing two different sugar-associated promoters.
In order to achieve this improvement, we have built 2 plasmidic constructs harboring 4 gene insert slots. This would allow the user to control the expression of the gene of interest in time with sugar of 4 different genes at a time.
As our results show, our construct behaves according to the hierarchy described in Guy Aidelberg et al’ paper (1). We notice that in the double insert construct pRIB is active in the presence of ribose but not in the presence of arabinose, however pARA is activated in the presence of both sugars.
In fact, arabinose being higher up in the hierarchy than ribose, the promoter associated with arabinose (pARA) is activated in the presence of both sugars (Arabinose and Ribose). However the ribose-associated promoter, in the double construct, in the presence of arabinose is not activated, as our last results confirms it. For our tool we would simply need to modify this one detail in the natural mechanism found in E.Coli. We have thought of an inhibition system using miRNA would be placed under the control of pRIB and that would inhibition activation of pARA when pRIB is activated . This can be explained by the action of the arabinose promoter, which is higher in the hierarchy and exerts its inhibitory action on the promoters located lower in the hierarchy. In order to solve what is a problem in our project, we thought of using miRNAs whose action would be under the influence of the pRib promoter. To do this, it would be sufficient for the PRib promoter to be activated so that miRNAs can inhibit the activation of the pAra promoter and thus cancel its effect on the pRib promoter.
We found an inhibition system that consists in using different miRNA placed under the control of one of our promoters. The miRNA could target the 5’UTR region of a specific mRNA for instance. The sugar-associated promoter would be in control of the gene of interest as well as 3 miRNA targeting the sequences of the mRNA produced by the other sugar-associated promoter. Through this technique, we can cancel the cross-activation of our promoters by preventing the production of other proteins.
In fact both promoters are activated in the presence of their associated sugar. In simple insert constructs and double insert constructs . You can visit our results on our wiki.
We were not able to confirm the functionality of one of our promoters, pSRL, making one of our plasmids constructs unfunctional. In the future we would like to redesign this promoter so that we would obtain 2 functional double insert constructs.
(1) G. Aidelberg, B. D. Towbin, D. Rothschild, E. Dekel, A. Bren, and U. Alon, “Hierarchy of non-glucose sugars in Escherichia coli,” BMC Syst. Biol., vol. 8, p. 133, 2014.