The SAINTS: Saint Andrews iGEM neuralizes threatening staphylococus. (360G-Wellcome-091959_Z_10_Z)

We will build an E. coli that can detect and kill S. aureus. The project will consist of three steps (Fig 2): detection, destruction, and enhancement. First, we will build E. coli to signal detection of AlP by producing green fluorescent protein (GFP). We will use the BioBrick BBa_1746220 created by Cambridge in 2007 to detect group I AlP in E. coli; Cambridge did not succeed in verifying this BioBrick (igem.org), thus we will first "debug" it to create a working copy. In particular, we note that BBa_l746220 does not include SarA, a general transcription factor believed to act in concert with AgrA to activate promoters P2 and P3 (Novick 2003). We will explore addition of SarA to the BioBrick, as well as other refinements, such as including the entire P2 to P3 promoter regions (Novick 2003). We will pair this with GFP (BioBrick BBa_K203103) to cause fluorescence upon detection of AlP. We will then create our S. aureus destroying E. coli by replacing the GFP with the "Kamikaze" BioBrick BBa_K284022 created by UNICAMP-Brazil in 2009: BBa_K284022 creates lysosyme, thus killing the cell and any nearby bacteria, and has been verified to work (iGem. org). Finally, we will enhance the sensitivity of both these constructs by inserting an E. coli quorum sensing loop in between the AlP detector and the output (e.g. verified BioBrick BBa_l15030, UT Austin 2005): this serves to amplify the signal from AlP to produce greater output (GFP or lysosyme) per cell and recruit other nearby E. coli.