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From 2012.igem.org

Pathogenic Cell Surface Biosensor

Overview

Current methods of water-borne pathogen detection require expensive equipment that often isn't modular, high throughput, or accessible to developing countries. One of the projects this year's ASU iGEM undertook was building a split-enzyme engineered fusion protein consisting of the Magainin peptide and the split alpha and omega fragments of beta-galactosidase.

Magainin

Magainins^{[http://www.chemicalbook.com/ChemicalProductProperty_EN_CB1199778.htm]} are a class of proteins found in the skin of Xenopus laevis^{[http://en.wikipedia.org/wiki/Xenopus_laevis]} - the African clawed frog - that contain antimicrobial properties. Both Magainin 1 and 2 are closely related peptides of 23 amino acids each and only differ by two amino acid substitutions. These two peptides have broad spectrum antimicrobial activity, including gram-positive and gram-negative bacteria, viruses, protozoa, yeasts and fungi, and are hemolytic and cytotoxic to cancer cells^{[http://www.pnas.org/content/85/3/910.full.pdf]}. The mechanism for antimicrobial activity in Magainin 1 and 2 involves the disruption the plasma membranes of target cells via electrostatic interactions between the negatively charged phospholipid bilayer and the positively-charged amino-terminus of the Magainin peptide.

The design of the biosensor took advantage of the electrostatic interaction between the Magainin-1 peptide and the lipid bilayer of bacteria to design a bacterial biosensor that confers a signal based on the binding of multiple Magainin peptides to a bacterial membrane (Figure A).

Biosensor Design Concept

Figure A depicts the Magainin-Linker-Split Beta-galactosidase biosensor design concept. In close proximity, the two subunits of beta-galactosidase, Alpha and Omega, interact to form a complete, fully-functional unit. Because Magainin peptides congregate and form a toroidal pore^{[http://www.springerlink.com/content/tg476537mk033550/]} (shown below) when binding to the membranes of bacteria, the close proximity of the linked split beta-galactosidase units can theoretically be achieved.

Progress Map

Results

Project progress has been completed up to cloning in the pET29 expression vector. Three of the four fragments of the split beta-galactosidase - omega, alpha-1, and 1-omega - have been isolated from the beta-galactosidase plasmid. Alpha-1 and 1-Omega, which have been isolated, complement each other to create a functional beta-galactosidase unit. Transformation of the alpha fragment in BL21(DE3) strains naturally coding for the Omega fragment have shown complementation in vivo, indicating that the split beta-galactosidase reporter system functioned as desired.

Expression of the full Magainin-Linker-Bgal construct in BL21(DE3) strains will continue post-Regional jamboree.