Team:KAIST Korea/Project Background
From 2012.igem.org
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+ | As the name tells us, our new biobrick part <span style="color:#aa0000;"><b>FlipFlop(BBa_K907002 and BBa_K907003)</b> </span> is a noble device which can generate binary signal.</br></br> | ||
+ | <span id="little">This part is composed of three sub-parts.</br></br></span> | ||
+ | <div style="border:2px solid;padding:0px 20px 0px 20px;"> | ||
+ | <ol style="padding:10px 0px 10px 30px;"> | ||
+ | <li><b>Promoter</b> : <a href="http://partsregistry.org/Part:BBa_J23119">BBa_J23119</a></li> | ||
+ | <li><b>RBS</b> : <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a></li> | ||
+ | <li><b>att sites</b> : Recognition site for <a href="http://partsregistry.org/Part:BBa_K907000">BBa_K907000</a>(Mycobacteriophage Bxb1 integrase).</li> | ||
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+ | <div style="color:#0000aa;font-size:25px;font-weight:bold;position:relative;top:-12px;"><span style="background-color:#fff;padding:0px 10px 0px 10px;">Signal 0</span></div> | ||
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+ | <img style="width:570px;" src="https://static.igem.org/mediawiki/2012/1/10/KAIST_Proj_Overview2.png"/> | ||
+ | </div> | ||
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+ | Initially, the FlipFlop promotes the transcription and translation of down-stream gene Protein A according to its promoter orientation. | ||
+ | <div align='center'> | ||
+ | <br><img style="width:340px;" src="https://static.igem.org/mediawiki/2012/a/ae/KAIST_Proj_Overview3_2.PNG"/> | ||
+ | </div> | ||
+ | When <a href="http://partsregistry.org/Part:BBa_K907000">BBa_K907000</a>(Mycobacteriophage Bxb1 integrase, Bxb1_Int) recognizes and inverts the sequence flanked by attB and attP sequences, promoter orientation is reversed, leaving recombined attL and attR sequences. | ||
+ | </div> | ||
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+ | <div style="border:2px solid #CC0000;padding:0px 20px 20px 20px;"> | ||
+ | <div style="color:#CC0000;font-size:25px;font-weight:bold;position:relative;top:-12px;"><span style="background-color:#fff;padding:0px 10px 0px 10px;">Signal 1</span></div> | ||
+ | <div align='center'> | ||
+ | <img src="https://static.igem.org/mediawiki/2012/1/1e/KAIST_Proj_Overview4.png"/> | ||
+ | </div> | ||
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+ | Then this device promotes the transcription and translation of upstream gene(must be designed in reverse orientation at construction step), or Protein B(Signal 1) in Figure 4.</br></br> | ||
+ | <div align='center'> | ||
+ | <img src="https://static.igem.org/mediawiki/2012/4/40/KAIST_Proj_Overview5.png"/> | ||
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- | </ | + | Also, the signal can be restored to its initial state when <a href="http://partsregistry.org/Part:BBa_K907000">BBa_K907000</a>/ <a href="http://partsregistry.org/Part:BBa_K907001">BBa_K907001</a> (Mycobacterio-phage Bxb1 excisionase, Bxb1_Xis) complex recognizes and inverts the sequence flanked with attL and attR sequences back to original state.</br> |
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- | + | <div style="color:#0000aa;font-size:25px;font-weight:bold;position:relative;top:-12px;"><span style="background-color:#fff;padding:0px 10px 0px 10px;">Signal 0</span></div> | |
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<div align='right'><a href="#top">▲Back to the top</a></div> | <div align='right'><a href="#top">▲Back to the top</a></div> |
Revision as of 19:37, 26 October 2012
2012 KAIST Korea
Mail : kaist.igem.2012@gmail.com
Twitter : twitter.com/KAIST_iGEM_2012
Facebook : www.facebook.com/KAISTiGEM2012
Project : Overview
- Promoter : BBa_J23119
- RBS : BBa_B0034
- att sites : Recognition site for BBa_K907000(Mycobacteriophage Bxb1 integrase).
Signal 0
Signal 1
Signal 0
What is bFMO?
Bacterial flavin-containing monooxygenase(bFMO) converts indole into isatin, which is then sequentially catalyzed into indigoid compounds emitting indigo color.
Because the enzyme utilizes the primary metabolite, we can easily notice the enzyme is working well or not. For the following experiments, engineered bFMO gene from Methylophaga sp. Strain SK1 is kindly provided by Duhee Bang from Yonsei University, Republic of Korea.
https://2012.igem.org/Team:KAIST_Korea/Project_Results
Working scheme of Our Design
At its initial state (while signal = 0), downstream gene of promoter, LuxI, produces AHL(N-Acyl homoserine lactone). Separated from FlipFlop device, LuxR gene is transcribed under the control of constitutive promoter. Generated LuxR molecules and AHLs form complex to initiate the transcription of Bxb1 integrase, positioned at the downstream of pLuxR promoter. Then, Bxb1 integrases specifically bind to attB and attP sequences to generate signal 1.
At its inverted state(while signal = 1), bFMO gene of upstream sequence can be transcribed and translated. So that bio-indigo starts to be produced. Upper genes of bFMO, AHL-Lactonase and Bxb1 Excisionase are expressed in order, degrading AHL-LuxR complexes and forming Bxb1 integrase-excisionase complexes, respectively. Finally, Int-Xis complexes bind to attL and attR sequences and invert the promoter into its original orientation, regenerating attB and attP sequences.