Over view

According to “NEWS”, we inserted “Genomic Pythagorean Device”, which we designed in a genome of E.coli.

“Pythagorean Devices” are very familiar to many Japanese people because Japanese famous educational TV program named “Pythagorean Switch” introduces a lot kind of them. They are known as "Rube Goldberg machines" in the US. Pythagorean Devices are deliberately over-engineered or overdone systems that perform very simple task in very complicated process and they usually include some chain reactions. We can enjoy this complicated process. In this year, we tried to design and make the device which is like “Pythagorean Device” in a genome of E.coli. We are very interested in site specific recombination systems in bacteria, so we used a lot kind of site specific recombination enzymes in our device. They were useful to make the process of this device more complicated.

↓What is “Pythagorean Device”? –Like this!

How dose it work?

“Genomic Pythagorean Device” is constructed by following 4 fragments.

We made these fragments by over rap extension PCR and inserted in a genome of E.coli according to the following figures.

We inserted them in a genome by lambda bacteriophage recombination system, “RED”. Then, how does this device work?

1. A large quantity of lacI is expressed by lacIq gene.
2. LacI represses plac.
3. By adding arabinose, pBad is activated and site specific recombination enzyme, HbiF comes to be expressed. 　
4. Because of HbiF, inversion happens between the site of IRL and IRR. And then　site specific recombination enzyme named Flp comes to be expressed.
5. Site specific recombination happens and the sequence between the 2 site of FRT is left off by Flp.
6. Repression of plac becomes off by deleting of lacIq gene and then β-gal comes to be expressed.
7. Surrounded E.coli in the form of Japanese character of “ピ” on the plate which x-gal was added in beforehand.
8. The form of the character of “ピ” comes to blue.

Retrieved from "http://2013.igem.org/Team:TMU-Tokyo/Project/Pythagorean_Device"