For the 2011 St Andrews iGEM Team project, we are creating an intracellular Escherichia coli “kill switch” that functions differently from any found in nature. This kill switch is a tool, one which we believe will have application within many areas of biology.
Our kill switch is designed by inserting an antimicrobial peptide (AMP) gene into E. coli. The AMP in question is protegrin-1, an 18 amino acid residue peptide first identified in porcine leukocytes (NMR structure pictured to the right). Protegrin-1 has high microbicidal activity against E. coli (gram-negative), N. gonorrheae (gram-positive), and HIV-1 (lipid-coated virus), amongst several other bacterial and virion species.
Protegrin-1’s secondary structure is a β-sheet conformation including a β-hairpin turn, which allows it to imbed itself into the phospholipid bilayer and disrupt bacterial cell walls by creating pores within the membrane (Lam et al., 2006). Pore formation between the cytoplasm and the extracellular space inhibits the cell’s ability to control ion movement, cytosol make-up, as well as its own structural integrity. The damage caused by this lack of control inevitably leads to cell death.
The antimicrobial activity of protegrin-1, including the action of pore formation, provides us with a wealth of potential applications for this kill switch. The one we chose to focus on is the idea of drug delivery. Pore formation within the membrane will cause the highly pressurized cell to lose vast amounts of its cytosol into the extracellular matrix. If a “drug” of some nature is present within the cell, it too will be exported along with the rest of the intracellular contents. If the drug needs to be delivered to say, the intestines, the natural biospecificity of E. coli to colonize the gut could improve on the broad-spectrum drug delivery method of pill digestion. We have explored this, and other potential applications, in our Uses section.
Lam et al. “Mechanism of Supported Membrane Disruption by Antimicrobial Peptide Protegrin-1”. The Journal of Physical Chemistry B, Vol. 110, pg. 21282 - 21286. Published 2006. Paper.