Team:HZAU-China/Construction

Our rewirable circuit includes three systems: input system, processing system and output system.

For input system, which is supposed to prevent the leaky expression of Cre protein, it is composed of taRNA, crRNA, Cre protein and corresponding promoters. We used the standard method of biobrick connection to generate the input system. During the process, of course, standardized crRNA cannot be found in the Plate Kits and it is tough to get and restandardize, so, we decided to synthetize this small segment by PCR (as shown in protocol). Luckily, we found the optimal condition and get the crRNA which is standardized, and finally finished the construction of input system.

For processing system, which is one of the toughest parts in our project, one is composed of three repressor genes---LacI, TetR and cI, their corresponding promoters and two recombination sites; another is composed of three genes related to generation and degradation of AHL molecule---AiiA, LuxR, LuxI, their corresponding promoters and two recombination sites. Due to design of the circuit, we have to connect two genes reversely to put their corresponding promoters between lox71 and lox66. To this end, we came up with a strategy that one segment is digested by EcoRI and SpeI, another segment is digested by EcoRI and SpeI and the vector is digested by XbaI and PstI, and finally do a ligation.

As to output system, we design two methods to detect whether our processing system works or not. One of them is fluorescent protein, we used mCherry protein and Cyan Fluorescent Protein to detect expression of two promoters simultaneously; due to the half-life of fluorescent protein and its quenching, which may interrupt the result of repressilator or positive-feedback loop, we design another detection device---fluorescent RNA, termed Spinach, which is a complex of RNA aptamers and fluorophore. We synthetized the fluorophore by ourselves through chemical experiments and we synthetized the RNA aptamers which is standardized by company.