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.

Objectives: Bacterial assembly is aimed to be achieved based on the mechanism of site-specific recombination systems, So that the expensive reagent as well as the laboring tasks could be saved in gene cloning experiments.

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?
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 integraded into one circular DNA, and 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 preserves the killer gene ccdB, which is under the control of another inducible promoter. When induced, the promoter will express CcdB so that cells containing CcdB will be killed. In order to link 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.