Team:BIT/Composite Part
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Composite Part
Integrase is able to identify a different specific sequences, called attP (on phage) and attB (in the host). After restructured and inserted in the phage, attP and attB will be flipped and converted into a pair of sites called attL and attR, which are no longer substrates for the recombinase. Using attP and attB sites, deliberately construct a modified DNA sequence, in this way you can "cheat" recombinase, so that the DNA sequence occur to irreversible reversal. The part contains the FimE integrase as well as its recognition sites attP, attB and BxB1 integrase as well as its recognition sites IRR, IRL. We use FimE and BxB1 to control the generation of GFP. In addition, the inhibition mechanisms of tetR protein on ptet promoter and LacI protein on plac promoter are used to control the expression of FimE and BxB1. If to add a certain amount of tetracycline to the inoculum, tetracycline will combine with TetR protein, the inhibition of TetR on ptet will disappear, and the downstream genes of FimE and BxB1 integrase can express. They will respectively act on the recognition sites behind the constitutive promoter, so that the site occurs 180 ° reversal and the original positive terminator becomes the reverse terminator, with the loss of the role of termination of transcription. Finally, the GFP can be expressed. This system can verify the function of integrase, also can be used as a detection mechanism to detect the presence of tetracycline, thus providing the theoretical basis for the development of our formal experiment. Using this mechanism can detect more material by changed the promoter and the promoter’s inhibitors.
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Beijing Institute of Technology,
No. 5 South Zhong Guan Cun Street,
Haidian Beijing 100081, P. R. China
Twitter : @igem_BIT
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Website : http://www.bit.edu.cn
