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Team:St Andrews/switch
From 2011.igem.org
The Kill Switch
The aim of our 2011 iGEM project is to create an E. coli kill switch using intracellular antimicrobial peptide (AMP) production.
[Diagram of biobrick (K206000 + J61101 + gene + B0015)]
The pBAD promoter is found in nature governing the E. coli arabinose operon, responsible for the breakdown of the sugar arabinose into D-xylulose-5-phosphate. This promoter is induced by the binding of L-arabinose to the AraC promoter region (further details on promoter structure and regulation can be found on our Modelling page). As a faster rate of protegrin-1 production would benefit our project by allowing for a wider range of potential applications, we chose to use the pBAD strong promoter (K206000), which is a mutagenized form of pBAD that induces transcription at a lower arabinose concentration and has a higher maximum expression. pBAD strong is only induced in the presence of arabinose, which is not naturally produced by the cell, making it a very stable promoter to work with and greatly reducing the chance of accidental gene activation.
Our ribosome binding site (RBS) is classified as a “strong” binding site (J61101), meaning that the ribosome will very readily bind to the mRNA sequence, allowing for translation to occur quickly after transcription has finished. We chose this RBS in order to help promote the rapid production of protegrin-1. Our terminator is a double terminator (B0015), standard in iGEM to decrease the chance of over-transcription by DNA polymerase.
Intracellular AMP production? Dr. Coote. etc.
Experiment Planning
We devised two sets of proof-of-concept experiments: one to ensure that our kill switch was functioning, and the other to test how it would work within a drug delivery context.
As our kill switch functions by killing cells, an obstacle we came across was how exactly to measure for cell death. With inspiration from the 2010 Berkley iGEM team (Berkely, 2010), we realized that we could use light absorbance as a proxy for cell death, with the idea that an intact cell will absorb more light than one that has burst due membrane disruption. An increase in optical density would correlate to cell growth, while a decrease would correlate to cell death.
Below are copies of our protocols created to test our AMP gene:
Kill Switch Proof of Concept Protocols (THIS NEEDS EDITING)
Drug Delivery Proof of Concept Protocols (THIS NEEDS EDITING)
Experiment Results
Characterization Data
pBAD Strong/Arabinose Characterization Protocols (THIS NEEDS EDITING)
