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)

"Genomic Kill Switch"

In Biobricks that are inserted in genome, horizontal gene transfer rate is lower than Biobricks are shipped by plasmids. This point is very important merit at the aspect of biosafety.

However, BioBricks which are in genome have stable hereditary too. This point may become demerit at the aspect of biosafety. If the organisms which have some BioBricks are in their genome released to natural environment, genetically modified materials would remain stably in natural environment.

In order to solve these problems, we designed “Genomic Kill Switch” as our future plan. The switch changes E.coli into arabinose requirement.

This device has bacteriophage p22 cII repressor in the downstream of pBAD promoter which is activated by L-arabinose. And BBa_K124017 (which has Bacteriophage Lysis Cassette S105, R, and Rz) are placed in the downstream of p22 c II regulated promoter. According to this device, BBa_K124017 expression is repressed by p22 cII only when arabinose presents in environment. It is really difficult to maintain this condition in natural environment, so if the E.coli which have the Genomic kill switch released by accident, they can’t live any longer. Therefore, this switch is able to prevent to spread genetically modified materials.