Difference between revisions of "Team:Pasteur Paris/Design"

To express the degradation pathway in E.coli we decided to assemble the different genes into one operon. One question remained. What is the gene combination that will yield the bacteria with the most efficient metabolism? The location of a gene and the gene combination are critical point for the efficiency of the system. Our system is composed of 13 genes, which is equivalent to 6 billion random gene combinations.
We assumed that the genes coding for enzymes that are close in the metabolic pathway shouldn't be too much separate. Therefore we identified
4 clusters. To automate the mixing of the gene we used site-specific recombination by means of the Cre/lox we went a step further by identifying
5 self-recombining lox sequences. By assigning 1 specific lox sequence to one cluster and placing the last self-recombining between each cluster
we are able to change the position of each gene inside it's cluster and shuffle the cluster inside the operon. The promoter as well as the terminator
was put outside this combinatorial system to avoid the change of their position.
This organisation allowed us to reduce the number of combination down to 3552.
We thought to assemble the whole operon in two times. First we would assemble each cluster individually using the Gibson assembly method. This method allows assembling gene fragments carrying overlaps of about 25 bp lengths at each end.
We ordered as gene fragment the cluster-specific lox sequence extended with the overlapping sequences. We added at the beginning and at the ending of the cluster a non-coding nucleotide sequence for the purpose to PCR amplify the well-assembled construct.
After this first step we expected products ranging from 2 up to 6 kb (Fig.1).

At this stage we had to assemble the 4 clusters into a plasmid. We decided to switch from the Gibson assembly method to the Yeast assembly method, which is more accurate during replication. To perform yeast assembly the fragments that have to be assembled are required to display minimum 250 bp overlaps. Those fragments are then co-transformed in S.cerevisae with an open plasmid, in our case a shuttle plasmid.
In presence of this fragments yeast recognise those fragments as broken DNA and triggers its repair mechanisms. Homologous recombination is performed between the overlapping regions resulting after the resolution of the Holliday junction in an uncut plasmid harbouring our degradation operon. The 250 bp overlaps were ordered as gene fragments. These fragments contain the overlap of the previous cluster followed by the fifth self-recombining lox sequence and finished by the overlapping region of the following cluster.
The final assembly is framed with the BioBrick prefix and suffix, which will be used to insert our operon in pSB1C3 (Fig. 2).