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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.
Structure
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.
How does it work?
Protegrin-1 is an antimicrobial peptide (AMP) first derived from porcine leukocytes. These peptides are part of the innate immune system and function by attacking the membranes and intracellular processes of invading bacteria. Protegrin-1 functions to protect the body against non-host cells by integrating itself into the phospholipid bilayer of prokaryotic bacteria, destabilizing the membrane and causing pore formation (cite). These pores inhibit the cell’s ability to control transmembrane ion and water movement, resulting in cell death via either osmosis or cytosol loss.
The idea behind our kill switch would be to induce bacteria to produce AMPs intracellularly, and allow these peptides to integrate into the membrane. As the concentration of AMPs builds, pores will form in the membrane, inevitably leading to cell death.
Antimicrobial peptides are highly conserved within evolution and can be found throughout all classes of life. This is in part due to the fact that mutating to resist AMPs is near impossible. These proteins attack bacterial membranes, meaning that multiple chance non-detrimental mutations across multiple genes controlling phospholipid bilayer structure would be required in order to resist AMP interactions. The chances of these mutations occurring within a single replication are astronomically small. This means that the use of protegrin-1 as the functional aspect of our kill switch will never lead to the creation of an AMP-resistant strain of E. coli.
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.
Utilizing Biospecificity
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. Our first attempt was devised with inspiration from the 2010 Berkeley iGEM team (Berkeley, 2010), where they used 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. Weeks 6-9 were spent testing varieties of the protocol below.
Kill Switch Proof of Concept Protocols (THIS NEEDS EDITING)
Unfortunately, what we had not foreseen was the digestion of arabinose by the naturally occurring arabinose operon, providing energy for the cells to grow. These newly grown cells replaced the cells lost to protegrin-1 production and our absorbance kept increasing, masking the cell death we hoped to see via kill switch activation. We tried to integrate this into the experiment by measuring the rates of growth at various arabinose concentrations compared to a no-arabinose control, but we decided that the data was not sufficient to show kill switch activity.
In Week 10, with the iGEM Jamboree looming, we decided to view our E. coli under a light microscope in the hopes to watch our kill switch functioning. This inspired the idea to use fluorescent dyes to distinguish between live and dead cells, and then measure fluorescence in the same plate reader we used for measuring absorbance. We purchased the LIVE/DEAD Baclight Bacterial Viability kit, which functions via a mixture of two dyes. The first, SYTO 9, is absorbed by all cells regardless of whether they are alive or dead, and fluoresces green (at 530 nm) when excited (with 485 nm). The second, propidium iodine, is only absorbed by cells with damaged membranes. Propidium iodine competes with SYTO 9, fluorescing red (at 630 nm) when excited (with 485 nm) and reducing SYTO 9's fluorescence within that cell. Further details of how the LIVE/DEAD Baclight Bacterial Viability kit works can be found here.
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 doesn't
pBAD Strong/Arabinose Characterization Protocols (THIS NEEDS EDITING)
