Team:KAIST Korea/Project Background

From 2012.igem.org

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<img src="https://static.igem.org/mediawiki/2012/9/9f/KAIST_Proj_Overview.png"/></br></br>
<img src="https://static.igem.org/mediawiki/2012/9/9f/KAIST_Proj_Overview.png"/></br></br>
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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 as its name implies.</br></br>
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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>
<span id="little">This part is composed of three sub-parts.</br></br></span>
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The FlipFlop, initially promotes the transcription and translation of down-stream gene, Protein A in the Figure 2, according to its promoter orientation.  
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Initially, the FlipFlop promotes the transcription and translation of down-stream gene Protein A according to its promoter orientation.  
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<br><img style="width:340px;" src="https://static.igem.org/mediawiki/2012/a/ae/KAIST_Proj_Overview3_2.PNG"/>
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When <a href="http://partsregistry.org/Part:BBa_K907000">BBa_K907000</a>(Mycobacteriophage Bxb1 integrase, Bxb1_Int) recognizes and inverts the sequence flanked with attB and attP sequences, promoter orientation is reversed, leaving recombined attL and attR sequences.  
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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.  
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Using the FlipFlop device, we can generate biological machine that operating the metabolism as we programmed. We decided to call the system as <span style="color:#aa0000;"><b>Auto-regulating FlipFlop</b></span>. To demonstrate the idea, we applied the device to control bio-indigo production, which is catalyzed by bacterial flavin-containing monooxygenase(bFMO).</br>
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Using the FlipFlop device, we can generate biological machine that controls the metabolism as we intended. We decided to call the system as <span style="color:#aa0000;"><b>Auto-regulating FlipFlop</b></span>. To demonstrate the idea, we applied the device to control bio-indigo production, which is catalyzed by bacterial flavin-containing monooxygenase(bFMO).</br>
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<span style="font-size:20px;font-weight:bold">What is bFMO?</span></br></br>
<span style="font-size:20px;font-weight:bold">What is bFMO?</span></br></br>
<img src="https://static.igem.org/mediawiki/2012/c/c8/KAIST_Overview_2nd_3.png" style="float:right; padding: 10px 10px 10px 10px;"/>
<img src="https://static.igem.org/mediawiki/2012/c/c8/KAIST_Overview_2nd_3.png" style="float:right; padding: 10px 10px 10px 10px;"/>
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Bacterial flavin-containing monooxygenase(bFMO) convert indole, which is produced from primary metabolite of organisms, into isatin, sequentially catalyzed into indigoid compounds that express indigo color.
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Bacterial flavin-containing monooxygenase(bFMO) converts indole into isatin, which is then sequentially catalyzed into indigoid compounds emitting indigo color.
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</br>Because the enzyme utilizing 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.
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</br>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.
<img src="https://static.igem.org/mediawiki/2012/b/b9/KAIST_Overview_2nd_4.png" style="padding: 30px 10px 10px 10px;"/>
<img src="https://static.igem.org/mediawiki/2012/b/b9/KAIST_Overview_2nd_4.png" style="padding: 30px 10px 10px 10px;"/>

Revision as of 05:52, 26 October 2012

KAIST Korea 2012 iGEM

Project : Overview




As the name tells us, our new biobrick part FlipFlop(BBa_K907002 and BBa_K907003) is a noble device which can generate binary signal.

This part is composed of three sub-parts.
  1. Promoter : BBa_J23119
  2. RBS : BBa_B0034
  3. att sites : Recognition site for BBa_K907000(Mycobacteriophage Bxb1 integrase).


Signal 0

Initially, the FlipFlop promotes the transcription and translation of down-stream gene Protein A according to its promoter orientation.

When BBa_K907000(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.


Signal 1

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.


Also, the signal can be restored to its initial state when BBa_K907000/ BBa_K907001 (Mycobacterio-phage Bxb1 excisionase, Bxb1_Xis) complex recognizes and inverts the sequence flanked with attL and attR sequences back to original state.


Signal 0







Using the FlipFlop device, we can generate biological machine that controls the metabolism as we intended. We decided to call the system as Auto-regulating FlipFlop. To demonstrate the idea, we applied the device to control bio-indigo production, which is catalyzed by bacterial flavin-containing monooxygenase(bFMO).


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.



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