Difference between revisions of "Part:BBa K902084"

Latest revision as of 20:08, 28 October 2012

Prha-B0034-S7(active)

In this part, [http://2012.igem.org/Team:Calgary iGEM Calgary 2012] implemented the rhamnose promoter as part of their active kill switch. P_rha, which is repressed by glucose and induced by rhamnose, is being used to express S7 micrococcal nuclease. This is an active killswitch where upon induction with rhamnose, the nuclease is produced, resulting in cell death. Conversely, when glucose is added, the promoter is repressed, causing limited expression of the nuclease gene, resulting in the cells being able to grow.

We characterized this circuit using differing concentrations of glucose and rhamnose. The results show that glucose causes deactivation of the killswitch whereas rhamnose results in its activation. The P_rha promoter was initially characterized via fluorescense using GFP. This data can be found here. Please see our [http://2012.igem.org/Team:Calgary/Project/Synergy Wiki] for more information!

Figure 1. P_rha-S7 construct demonstrating our kill switch in TOP10 wild type cells and glyA knockout cells. This demonstrates that our system is capable of being induced by the sugar rhamnose and repressed in the presence of glucose. There is no growth in rhamnose with our system as the RhaBAD operon has been deleted in the knockout strain we are using.

Note that the above figure shows redundancy of our kill system. S7 and the rhamnose promoter are the active component of this system. We combined this system with E. coli that are auxotrophic and dependent on glycine supplementation. While each of these elements alone were able to kill and control cells, the combination of these two mechanisms together were shown most effective.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K902084 short</partinfo>
-<html>This part the Prha promoter, which is repressed by glucose and induced by rhamnose upstream of <i>S7 micrococcal nuclease</i>. This is an active killswitch where upon induction with rhamnose, the nuclease is produced, resulting in cell death.  Conversely, when glucose is added, the promoter is repressed, causing no expression of the nuclease gene, resulting in the cells being able to grow.  We characterized this circuit using differing concentration of glucose and rhamnose.  The results show that with increasing concentrations of glucose, this killswitch is deactivated.
+In this part, [http://2012.igem.org/Team:Calgary iGEM Calgary 2012] implemented the rhamnose promoter as part of their active kill switch. [https://parts.igem.org/wiki/index.php?title=Part:BBa_K902065 P<sub>rha</sub>], which is repressed by glucose and induced by rhamnose, is being used to express [https://parts.igem.org/wiki/index.php?title=Part:BBa_K902019 <i>S7 micrococcal nuclease</i>]. This is an active killswitch where upon induction with rhamnose, the nuclease is produced, resulting in cell death.  Conversely, when glucose is added, the promoter is repressed, causing limited expression of the nuclease gene, resulting in the cells being able to grow.
-The Prha promoter was initially characterized via fluorescense using GFP.  This data can be found  <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K902066">here</a>. Please see our <a href="http://2012.igem.org/Team:Calgary/Project/Synergy">Wiki</a> for more information!</html>
-[[Image:Calgary Rha S7 Data.png|thumb|500px|center|Figure 1. pRHA-S7 construct demonstrating our kill switch in TOP10 wild type cells and glyA knockout cells. This demonstrates that our system is capable of being induced by the sugar rhamnose and repressed in the presence of glucose. There is no growth in rhamnose with our system as the RhaBAD operon has been deleted in the knockout strain we are using..]]
+We characterized this circuit using differing concentrations of glucose and rhamnose.  The results show that glucose causes deactivation of the killswitch whereas rhamnose results in its activation.
+The P<sub>rha</sub> promoter was initially characterized via fluorescense using GFP.  This data can be found [https://parts.igem.org/wiki/index.php?title=Part:BBa_K902066 here]. Please see our [http://2012.igem.org/Team:Calgary/Project/Synergy Wiki] for more information!
+[[Image:Calgary Rha S7 Data.png|thumb|500px|center|<i>Figure 1</i>. P<sub>rha</sub>-S7 construct demonstrating our kill switch in TOP10 wild type cells and glyA knockout cells. This demonstrates that our system is capable of being induced by the sugar rhamnose and repressed in the presence of glucose. There is no growth in rhamnose with our system as the RhaBAD operon has been deleted in the knockout strain we are using.]]
+Note that the above figure shows redundancy of our kill system. S7 and the rhamnose promoter are the active component of this system. We combined this system with <i>E. coli</i> that are auxotrophic and dependent on glycine supplementation. While each of these elements alone were able to kill and control cells, the combination of these two mechanisms together were shown most effective.