Team:Nantes/Design
The design of our project incorporates the elegant sugar-consumption machinery, naturally found in E.Coli.
We are basing our project on a hierarchy that naturally exists in the K12 MG1655
Escherichia Coli strand. This is in fact the strand that was used in the article that inspired our project.
In this article, the authors seem to have studied the activity of the different promoters
individually using a GFP (green fluorescent protein) reporter gene. We would like to go one step further and study the activity of these promoters simultaneously. Therefore
each promoter triggers the expression of a different fluorescent protein.
Our goal was to build two plasmids each containing two promoter/fluorescent gene couples.
Having 2 plasmids would allow us to add a toxin/antitoxin system that would significantly decrease the risk of a plasmid transfer between transformed bacteria and any other bacteria. We explain this system in further detail on our “Safety” page.
To do so we chose two plasmid matrixes that contain different antibiotic resistance genes
and origins of replication from different classes to lower the competition of replication
between the two different plasmids. These were provided by IDT.
We then selected four sugars of this sugar hierarchy : lactose, arabinose, sorbitol and ribose.
We chose these four specific sugars because that had the lowest score of cross-activation that
was observed in Aidelberg’s article.
So once we had chosen our plasmid matrixes and the promoters we were going to use, we needed to figure out which promoters we would pair up on the same plasmid. To do so we decided to put the sugars furthest away from each other in the sugar hierarchy together to limit cross activation so pLAC (lactose) with pSRL(sorbitol) and pARA (arabinose) with pRIB (ribose).
We then needed to associate each promoter with a fluorescent gene. We chose our reporter genes based on their excitation and emission spectra to be able to easily distinguish the activity of one promoter from another and to prevent unwanted activation of certain fluorescent genes.
And finally to complete the constructs we chose a strong Ribosome Binding Site (RBS) and two terminators from different classes from the iGEM part registry.
Our plasmids would look something like this :
Before building these plasmids however, we wanted to do an intermediate step by building one-insert plasmids, meaning that they would contain 1 promoter associated with 1 fluorescence gene.
We then proceeded to transforming XL1-blue E. Coli chemocompetent bacteria or top10 electrocompetent bacteria to amplify and add the methyl group to our plasmids. These plasmids are considere as bacteria DNA and not synthetic DNA. We would then proceed to transforming chemocompetent K12 bacteria with these plasmids.
We then proceeded to study the activity of these promoters in kinetic spectrophotometry and fluorometry in different conditions by varying the sugars present in the medium, the concentration of these sugars, the temperature and the pH. This data was used to build a model that allows the user to predict the amount of each specific sugar that needs to be added to the medium to have a specific gene expression at a certain point in time and for a certain duration in time.
The end goal would be to transform competent E.Coli of the K12 strand with our two plasmids so that they would contain all four of the promoters at the same time. The user could proceed to replacing the reporter genes by the gene of interest and could program gene expression in time by adding the specific concentration of sugar that was given by the model.
