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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.

Results & Progress Map

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 (shown below). 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.