More improved “Genomic Pythagorean Device”

We designed three kinds of devices which improved “Genomic Pythagorean Device”. The basic fragments constituting these three devices are same with the original device (Fig.1).

If you want to see more detailed explanation of our device which we made in this year, please go and look “Genomic Pythagorean Device"page.

・Improved device 1

In this year, we did PCR in order to check that lacI gene is deleted because of the presence of FRT. It was a good assay method but it takes time slightly. So first we designed the improved device 1 which is for easily assay of fragment 3-1 and 3-2. This device has GFP site in the downstream of plac. After lacI gene is deleted, repression of plac becomes off. Then GFP comes to be expressed so that we can confirm easily whether lacI gene is deleted or not. (Fig.2)

・Improved device 2

We designed more complicated device by using the site of lox66, lox71 and Cre recombinase. Lox66 and lox71 are two mutants of loxP site and Cre recombinase mediated recombination between them. When Cre recombinase is present, the DNA flanked by the two mutant loxP sites is inverted, forming one loxP and one double mutated loxP site. As the double mutated loxP site shows low affinity for Cre recombinase. So once Cre-mediated recombination happen, it becomes difficult to happen again. (Fig.3)

Then, we’ll describe that how this device works. This device has Cre recominase site in the downstream of plac. After lacI gene is deleted, repression of plac becomes off and Cre recombinase comes to be expressed. Then site specific recombination happen between the sites of lox66 and lox71 and the promoter which is in there turns over by inversion. By this result, GFP comes to be expressed and we can confirm easily whether lacI gene is deleted or not (Fig.4).

・Improved device 3

We designed more complicated device than improved device 2 by using attB/attP recombination system in addition of lox66, lox71 and Cre recombinase. Site specific recombinase named Int/Xis mediate recombination between the sites of attB and attP.

This device has Cre recominase site in the downstream of plac. After lacI gene is deleted, repression of plac becomes off and Cre recombinase comes to be expressed. Then site specific recombination happen between the sites of lox66 and lox71 and the promoter which is in there turns over by inversion. By this result, Int comes to be expressed. Then, site specific recombination happen because of the presence of Int and the plasmid which has the site of loxP and CI repressor is inserted in E.coli genome. CI repress the activity of pCI. Then Cre recombinase mediates the site specific recombination between the 2sites of loxP and CI repressor gene is deleted. By this result, repression of pCI becomes off and GFP comes to be expressed. (Fig.5)