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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.
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.
Concerns were raised about the effectiveness of intracellular AMP production. In nature, protegrin-1 attacks bacterial membranes from the outside by burrowing a hole into the outer leaf of the phospholipid bilayer. We were unsure of protegrin-1's ability to perform this action from the inside of the cell; specifically, whether or not it would interact with the inner sheet of phospholipids. Little research has been conducted on intracellular AMP production, so with no directly available answer, we decided to explore for ourselves. All the material we found provided no strong evidence that there was much structural difference between the two layers of prokaryotic membrane. Both layers have a mixture of zwitterionic and acidic phospholipids, the latter of which contain a negative charge that facilitates the electrostatic interactions between the AMP and the bilayer. Eukaryotes have almost no acidic phospholipids on their outer layer, and imbedded in between the lipids is cholesterol, which increases the stability of the phospholipids and impedes hydrophobic AMP interaction (in conjunction, these two traits provide the protection that stop AMPs from attacking host cells). So, as long as there are a sufficient concentration of acidic phospholipids to facilitate the interaction, the AMPs should be able to bind to either leaflet. We also contacted resident biomolecular scientist Dr. Peter Coote, who has authored several papers on AMPs, to provide advice on this subject. He confirmed our theory that acidic phospholipids were imperative for the interaction between bilayer and peptide to occur, and agreed that intracellular protegrin-1 production should, at least hypothetically, cause cell death.
Dr. Coote also provided us with gomesin, an AMP structurally analogous to protegrin-1, for use in testing our kill switch theory.
Promoter specificity for use around the body
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 Berkeley iGEM team (Berkeley, 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
The aim of a "characterization experiment" is to gather more data about the biobrick in question. Submitting a biobrick that has only been tested under a single condition
pBAD Strong/Arabinose Characterization Protocols (THIS NEEDS EDITING)
