Team:KAIST Korea/Project Background

From 2012.igem.org

(Difference between revisions)
Line 39: Line 39:
{
{
width:200px;
width:200px;
-
height:30px;
+
height:30px;
position:relative;
position:relative;
background:#f2f3f3;
background:#f2f3f3;
padding:5px 30px 5px 30px;
padding:5px 30px 5px 30px;
border:1px solid #f4f4f4;
border:1px solid #f4f4f4;
-
margin : 0 20px 0 20px;
+
margin : 0 20px 0 20px;
}
}
Line 97: Line 97:
#kaistcontent a {   
#kaistcontent a {   
-
    color:#5a5b5e;   
+
color:#5a5b5e;   
}   
}   
Line 143: Line 143:
</br>
</br>
<div align='center'>
<div align='center'>
-
<a href="#1st"><span id="tab">  How the Filp Flop works </span></a>
+
<a href="#1st"><span id="tab">  <b>How the Filp Flop works </b> </span></a>
-
<a href="#2nd"><span id="tab">How the Filp Flop can be applied</span></a>
+
<a href="#2nd"><span id="tab"><b>How the Filp Flop can be applied</b></span></a>
</div>
</div>
</br>
</br>
Line 228: Line 228:
</div>
</div>
-
</br></br>
+
</br></br>https://2012.igem.org/Team:KAIST_Korea/Project_Results
<div style="border:2px solid #000000;padding:20px 20px 20px 20px;">
<div style="border:2px solid #000000;padding:20px 20px 20px 20px;">
<span style="font-size:20px;font-weight:bold">Working scheme of Our Design</span></br></br>
<span style="font-size:20px;font-weight:bold">Working scheme of Our Design</span></br></br>

Revision as of 11:57, 19 October 2012

KAIST Korea 2012 iGEM

Project : Overview




Our new biobrick part FlipFlop(BBa_K907002 and BBa_K907003) is a noble device which can generate binary signal as its name implies.

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

The FlipFlop, initially promotes the transcription and translation of down-stream gene, Protein A in the Figure 2, according to its promoter orientation.

When BBa_K907000(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.


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 operating the metabolism as we programmed. 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) convert indole, which is produced from primary metabolite of organisms, into isatin, sequentially catalyzed into indigoid compounds that express indigo color.
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.


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.



Kaist Footer

Retrieved from "http://2012.igem.org/Team:KAIST_Korea/Project_Background"