Difference between revisions of "Part:BBa K4193026"
 
(One intermediate revision by the same user not shown)
Line 17: Line 17:
 
Figure1.The signal pathway of IP-mediated quorum sensing
 
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">
 +
</div>
 +
</body>
 +
</html>
 +
Figure3. Effect of different concentrations of IP on SSRE promoter induction.
 +
 +
To get a great insight for the protein in designed signal pathway, we used dry lab to make up for the defect we could not characterize the behavior of these proteins, such as AtCRE1_P, Ypd1_P and Skn7_P interacting with different subtypes of SSRE promoter.
 +
<html>
 +
<head>
 +
<meta charset="utf-8">
 +
 +
</head>
 +
<body>
 +
<div>
 +
<img src="https://static.igem.wiki/teams/4193/wiki/other/part/phophoylated-function-protein-changes.jpg">
 +
</div>
 +
</body>
 +
</html>
 +
Figure4.The behavior of proteins in designed signal pathway
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 12:08, 12 October 2022


QS system

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 get a great insight for the protein in designed signal pathway, we used dry lab to make up for the defect we could not characterize the behavior of these proteins, such as AtCRE1_P, Ypd1_P and Skn7_P interacting with different subtypes of SSRE promoter.

Figure4.The behavior of proteins in designed signal pathway Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1092
    Illegal EcoRI site found at 3483
    Illegal EcoRI site found at 5357
    Illegal EcoRI site found at 5682
    Illegal XbaI site found at 2581
    Illegal XbaI site found at 3040
    Illegal XbaI site found at 3124
    Illegal XbaI site found at 3487
    Illegal XbaI site found at 6643
    Illegal SpeI site found at 5194
    Illegal PstI site found at 5444
    Illegal PstI site found at 6143
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1092
    Illegal EcoRI site found at 3483
    Illegal EcoRI site found at 5357
    Illegal EcoRI site found at 5682
    Illegal SpeI site found at 5194
    Illegal PstI site found at 5444
    Illegal PstI site found at 6143
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1092
    Illegal EcoRI site found at 3483
    Illegal EcoRI site found at 5357
    Illegal EcoRI site found at 5682
    Illegal BglII site found at 523
    Illegal BglII site found at 880
    Illegal BglII site found at 1129
    Illegal BglII site found at 2065
    Illegal BglII site found at 2539
    Illegal BglII site found at 4398
    Illegal BamHI site found at 633
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1092
    Illegal EcoRI site found at 3483
    Illegal EcoRI site found at 5357
    Illegal EcoRI site found at 5682
    Illegal XbaI site found at 2581
    Illegal XbaI site found at 3040
    Illegal XbaI site found at 3124
    Illegal XbaI site found at 3487
    Illegal XbaI site found at 6643
    Illegal SpeI site found at 5194
    Illegal PstI site found at 5444
    Illegal PstI site found at 6143
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1092
    Illegal EcoRI site found at 3483
    Illegal EcoRI site found at 5357
    Illegal EcoRI site found at 5682
    Illegal XbaI site found at 2581
    Illegal XbaI site found at 3040
    Illegal XbaI site found at 3124
    Illegal XbaI site found at 3487
    Illegal XbaI site found at 6643
    Illegal SpeI site found at 5194
    Illegal PstI site found at 5444
    Illegal PstI site found at 6143
    Illegal NgoMIV site found at 6410
    Illegal NgoMIV site found at 6450
    Illegal NgoMIV site found at 6490
    Illegal NgoMIV site found at 6530
    Illegal AgeI site found at 6420
    Illegal AgeI site found at 6460
    Illegal AgeI site found at 6500
    Illegal AgeI site found at 6540
  • 1000
    COMPATIBLE WITH RFC[1000]