Team:Chiba/Circuit 2
From 2010.igem.org
Overall Circuit It consists of recognizing input system and memorizing input system. |
Control of circuit |
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. |
System_1)Recognizing input |
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 AHL come into Double Click Bacteria, generation of cI is stopped. Since repression of Plux starts working by the dimer which is a combination of AHL and LuxR. And then, cI is going to disappear, because all kinds of proteins are bound to be decomposed. If there isn't cI anymore, bacteria will have chance to shine. We can say cI recognizes the existence of input in this DNA sequence. |
System_2)Memorizing input |
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 AHL come into Double Click Bacteria, generation of cI is stopped. Since repression of Plux starts working by the dimer which is a combination of AHL and LuxR. And then, cI is going to disappear, because all kinds of proteins are bound to be decomposed. If there isn't cI anymore, bacteria will have chance to shine. We can say cI recognizes the existence of input in this DNA sequence. |
Second, there is a cI operator above gfp DNA sequence. |
Third, there is an AND Gate with T7ptag and supD.
On initial state, bacteria don't have supD.
As written above, when AHL is entered into bacteria, transcription of proteins
After washing AHL(means after 1st click), supD exist.
Moreover, if there is 2nd click, T7ptag is going to be decomposed again. |
Actually, there is a hybrid promoter(cI/T7) above gfp DNA sequence.
It is regulated by T7 and cI which work as activator and repressor, respectively.
On this promoter, repression is stronger than activation.
And also, it is low unregulated activation.
When 1st input is come in, cI recognizes the input and going to be decomposed.
But there isn't T7 yet, gfp cannot be generated, despite there isn't repressor.
After 1st input, AND gate remembers there was a click, and generation of T7 is stared.
But, gfp cannot be generated, because there is cI already.
When 2nd input is come, cI also recognizes the input and going to be decomposed..
If the 2nd input comes within time that the AND is working,
T7 exists in bacteria and decomposition of cI is finished, gfp is going to be generated.
As a result, bacteria shines with gfp and Double Click is completed.