Team:Chiba/Project

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Bacterial Double Click

If there are two-time input for limited time, bacteria shine.

Overall project

We’re inspired by double-click of computer’s mouse.

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.

Project Details

Requirement of Double Click

1. If there is a click only one-time, nothing happens.
2. Time of clicking button doesn't matter. It just depends on the number of click. One or two.
3. After a lot of time pass from the 1st click, it will return to the initial state.
4. Click two-time in specific limited time(We call this double click.), something occur.

Detail explanation

                    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 represses Plux, transcription of proteins under the
                    lux promoter is stopped.
                    So in our project,
                    CLICK controls transcription of overall DNA sequence.

                    Second,there is a cI operator above gfp DNA sequence.
                    cI works as repressor.
                    You can see cI DNA sequence is under lux promoter.
                    As AHL come into Double Click Bacteria, generation of cI is stopped.
                    Therefore, repressor of cI promoter disappears.
                    (Because the cause of repress(cI) which is kind of protein will be
                    decomposed.)
                    If there isn't cI, bacteria shine with gfp.
                    We can say
                    cI recognizes the existence of input in this DNA sequence.

                        Third, there is 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 T7 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 we can see bacteria shine with gfp.
                        On initial state(there isn't any click), bacteria don't have supD.
                        So there isn't any T7 polymerase and gfp cannot be genernated, in spite of being T7ptag in bacteria.
                        T7ptag which is a kind of mRNA is decomposed so fast.
                        However supD under lacI promoter is generated late.
                        Because, transcription of supD starts working after most of lacI are decomposed.
                        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,

Actually, there is a hybrid promoter(cI/T7) above gfp DNA sequence.
T7 works as activator and cI works as repressor to the promoter.

Part 3

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Results

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 represses Plux, transcription of proteins under the lux promoter is stopped. So in our project, CLICK controls transcription of overall DNA sequence.

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 So in our project,
 <b><font color="red">CLICK controls transcription of overall DNA sequence.</font></b>
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 |align="center"| [[Image:Chiba_LuxR.jpg‎|400px|left|frame]]
 |-
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+}
 <br><br><br>
 == Results ==