Part:BBa_K902084

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]