Project

The result of recombination is that the integrated prophage is flanked by two attachment sites but now they are slightly different: attL has the structure BOP' and attR has the structure POB'.

Cre-Lox recombination is a special type of site-specific recombination, which is often applied as a gene knockout tool.
Cre is a site-specific DNA recombinase, which can catalyse the recombination of DNA between specific sites, e.g. loxP in a DNA molecule. When cells that have loxP sites in their genome express Cre, a reciprocal recombination event will occur between the loxP sites. The double stranded DNA is cut at both loxP sites by the Cre protein. The strands are then rejoined with DNA ligase. The efficiency of recombination depends on the orientation of the loxP sites. For two lox sites on the same chromosome arm, inverted loxP sites will cause an inversion, while a direct repeat of loxP sites will cause a deletion event.

Lox P site
Lox P (locus of X-over P1) is a site on the Bacteriophage P1 consisting of 34 bp. There exists an asymmetric 8 bp sequence in between with two sets of palindromic, 13 bp sequences flanking it. The detailed structure is given below.

Our design

We have innovatively utilized the site-specific systems mentioned above to build a foolproof bacterial assembly system to future reduce the labor and cost involved in gene cloning experiments. We have designed three standardized vectors which perform as the donors, receptor vector respectively.

How do they work?
First, we define the Receptor as the vector that has already existed in the cell (E.coli.), and the Donor as the vector containing the desired gene that we intend to integrate into the Receptor. The gene circuits for these plasmids are illustrated below.

When the Donor vector carrying the gene of interest GENE1 was introduced to the E Coli which contains the Receptor vector, the site-specific recombination will occur between the attB1 site and the attP1 site, so that the two sequences will be integrated into one circular DNA.

The recombinant DNA then could be selected in the liquid culture containing both ampicillin and kanamycin. Then, under inducible conditions, Cre will be expressed and the recombined sequence will be divided into two separate plasmids; one will retain the desired gene 1, while the other will preserve the killer gene ccdB, which is under the control of another inducible promoter. Because the two plasmids have shared origin site, plasmid incompatibility will occur thus the two kinds of plasmids will be separated into different cells.

When induced, CcdB could be expressed so that cells containing CcdB will be killed.

In order to realize the linkage of GENE 1 with GENE 2, we will introduce the new plasmid containing the desired GENE2 to the survival cells, in which the plasmids containing GENE 1 will behave as the new Receptor plasmid. Very similarly recombination between the attB2 and attP2 and the cleavage between the two loxp  sites will be performed, and plasmids containing the linked GENE1 and GENE2 will be selected when the promoter expresses CcdB is induced.

The reason for us to use two sets of attB/attP specific sites is to avoid the combination within one molecule.