Difference between revisions of "Part:BBa K4193004"
 
(3 intermediate revisions by the same user not shown)
Line 5: Line 5:
 
Quorum sensing system related promoter
 
Quorum sensing system related promoter
  
−
<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
  
−
It is regulated by the quorum-sensing molecule IP, When the IP reaches a certain concentration, the promoter turns on the expression.
+
Cytokinin isopentenyladenine (IP), as quorum sensing molecule, is the product of ATP catalyzed by AtlPT4. When the signal molecule IP reaches a certain concentration, it activates the SSRE promoter and increases the expression of production gene controlled by SSRE promoter in our experiment, to better characterize SSRE promoter and quorum sensing circuit, we put eGFP gene at the downstream of SSRE promoter.
 +
<html>
 +
<head>
 +
<meta charset="utf-8">
 +
 +
</head>
 +
<body>
 +
<div>
 +
<img src="https://static.igem.org/mediawiki/parts/1/17/QS.jpg" style="width: 80%;">
 +
</div>
 +
</body>
 +
</html>
 +
Figure1.The signal pathway of IP-mediated quorum sensing
  
 +
We successfully introduced cytokinin-mediated quorum sensing circuit into the genome of Aureobasidium  melanogenum P16. To characterize this circuit and SSRE promoter, we added different concentration of IP and introduced eGFP at the downstream of SSRE promoter. We measured the fluorescence to figure out the dynamic regulation range.
 +
<html>
 +
<head>
 +
<meta charset="utf-8">
 +
 +
</head>
 +
<body>
 +
<div>
 +
<img src="https://static.igem.wiki/teams/4193/wiki/model/qs-result.jpg" style="width: 80%;">
 +
</div>
 +
</body>
 +
</html>
 +
Figure2.Gel electrophoresis results of P16 genome amplifying AtCRE1 gene (Lane1), transformed strain genome amplifying AtCRE1 (Lane2), P16 genome amplifying PTP2+eGFP (Lane3) and transformed strain genome amplifying PTP2+eGFP (Lane 4)
 +
 +
Different concentrations of IP can increase the expression of SSRE promoter. After rough statistical analysis, the expression intensity of SSRE promoter was significantly correlated with IP concentration.
 +
<html>
 +
<head>
 +
<meta charset="utf-8">
 +
 +
</head>
 +
<body>
 +
<div>
 +
<img src="https://static.igem.wiki/teams/4193/wiki/other/part/ssre-expression.jpg" style="width: 80%;">
 +
</div>
 +
</body>
 +
</html>
 +
Figure3. Effect of different concentrations of IP on SSRE promoter induction.
 +
 +
To obtain more subtypes SSRE promoter with various of dynamic regulatory ranges,we change the sequence to adjust the dissociation rate between SSRE promoter and Skn7_P protein and get more SSRE promoter with different dynamic regulatory ranges.
 +
<html>
 +
<head>
 +
<meta charset="utf-8">
 +
 +
</head>
 +
<body>
 +
<div>
 +
<img src="https://static.igem.wiki/teams/4193/wiki/other/part/different-ssre-expression-1.jpg" style="width: 80%;">
 +
</div>
 +
</body>
 +
</html>
 +
Figure4. Obtain more subtypes SSRE promoters by increasing the dissociation rate between SSRE promoter and Skn7_P protein
 +
 +
<html>
 +
<head>
 +
<meta charset="utf-8">
 +
 +
</head>
 +
<body>
 +
<div>
 +
<img src="https://static.igem.wiki/teams/4193/wiki/other/part/different-ssre-expression.jpg" style="width: 80%;">
 +
</div>
 +
</body>
 +
</html>
 +
Figure5.Obtain more subtypes SSRE promoters by decreasing the dissociation rate between SSRE promoter and Skn7_P protein
 
<!-- -->
 
<!-- -->
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 12:11, 12 October 2022


SSRE promoter

Quorum sensing system related promoter

Usage and Biology

Cytokinin isopentenyladenine (IP), as quorum sensing molecule, is the product of ATP catalyzed by AtlPT4. When the signal molecule IP reaches a certain concentration, it activates the SSRE promoter and increases the expression of production gene controlled by SSRE promoter in our experiment, to better characterize SSRE promoter and quorum sensing circuit, we put eGFP gene at the downstream of SSRE promoter.

Figure1.The signal pathway of IP-mediated quorum sensing

We successfully introduced cytokinin-mediated quorum sensing circuit into the genome of Aureobasidium melanogenum P16. To characterize this circuit and SSRE promoter, we added different concentration of IP and introduced eGFP at the downstream of SSRE promoter. We measured the fluorescence to figure out the dynamic regulation range.

Figure2.Gel electrophoresis results of P16 genome amplifying AtCRE1 gene (Lane1), transformed strain genome amplifying AtCRE1 (Lane2), P16 genome amplifying PTP2+eGFP (Lane3) and transformed strain genome amplifying PTP2+eGFP (Lane 4)

Different concentrations of IP can increase the expression of SSRE promoter. After rough statistical analysis, the expression intensity of SSRE promoter was significantly correlated with IP concentration.

Figure3. Effect of different concentrations of IP on SSRE promoter induction.

To obtain more subtypes SSRE promoter with various of dynamic regulatory ranges,we change the sequence to adjust the dissociation rate between SSRE promoter and Skn7_P protein and get more SSRE promoter with different dynamic regulatory ranges.

Figure4. Obtain more subtypes SSRE promoters by increasing the dissociation rate between SSRE promoter and Skn7_P protein

Figure5.Obtain more subtypes SSRE promoters by decreasing the dissociation rate between SSRE promoter and Skn7_P protein Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 239
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 239
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 239
    Illegal NgoMIV site found at 6
    Illegal NgoMIV site found at 46
    Illegal NgoMIV site found at 86
    Illegal NgoMIV site found at 126
    Illegal AgeI site found at 16
    Illegal AgeI site found at 56
    Illegal AgeI site found at 96
    Illegal AgeI site found at 136
  • 1000
    COMPATIBLE WITH RFC[1000]