Team:Chiba/Project

It doesn’t react by first input but react by second input. So we designed bacterial DNA sequence which works like it. Furthermore, we built time-limit in genetic sequence between the first and the second input like a computer mouse. Bacteria which have the DNA sequence can distinguish between the first input and the second one. Once in a while, bacteria might receive input which wasn’t intended. But it was the first input so bacteria don’t react. Even if there was input not intended, bacteria don’t react unless the second input is entered. Moreover, when the limit-time which controlled by the genetic system is passed, the system is going to return to the initial state. So we expect this DNA sequence will work as a safety device.

          First, we use AHL for input and regard injection of AHL as clicking.
          LuxR is generated constitutive.
          Injecting AHL to Double Click Bacteria brings dimer of AHL and luxR.              
          We take lux promoter repressed by the dimer.
          As the dimer binds Plux,
          transcription of proteins under the Plux is repressed.

          So in our project,
          CLICK controls transcription of overall DNA sequence.

          Second, there is a cI operator above gfp DNA sequence.
          If cI binds cI operator, transcription of gfp is stopped.
          You can see DNA sequence of cI, which a kind of repressor, is under the Plux.
          On initial state(there isn't any click), cI is generated normally.
          But after AHL come into Double Click Bacteria, generation of cI is stopped.
          And then, repressor of cI operator disappears.
          Because cI which is a kind of protein will be decomposed.
          If there isn't cI anymore, bacteria will shine with gfp.

          We can say
          cI recognizes the existence of input in this DNA sequence.

          Third, there is an AND Gate with T7ptag and supD.
          As you know, T7ptag becomes T7 polymerase in case of existing supD.
          T7 polymerase is translated to T7 protein
          And it works as activator of T7 promoter.
          There is a T7 promoter above gfp DNA sequence.
          If T7 have been generated once, the T7 promoter starts translating.                  
          And then we can see bacteria begin to shine.

          On initial state, bacteria don't have supD.
          Because, lacI represses the lac promoter above supD DNA sequence.
          So there isn't any T7 polymerase and gfp cannot be genernated,
          in spite of being T7ptag in bacteria.

          As written above, when AHL is entered into bacteria, transcription of proteins
          under the Plux is repressed.
          If there is click,
          transcription of T7ptag and lacI are stopped simultaneously.
          But T7ptag are decomposed first, and then lacI are decomposed with the
          difference in velocity of decomposition.
          T7ptag which is a kind of mRNA is decomposed so fast
          and lacI which is just a kind of protein is decomposed late.
          SupD under the Plac is generated after most of lacI are decomposed.
          So, in the clicking, there isn't any T7ptag as far as there is supD.

          After 1st click, supD exist.(supD is a kind of tRNA which is stable.)
          There isn't input anymore,
          promoter above T7ptag DNA sequence starts activating.
          As a result of reacting T7ptag and supD,
          the AND Gate is going to be available.
          In this case, the AND Gate remembers that there was a click.
          Moreover,