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From 2012.igem.org

Pathogenic Cell Surface Biosensor

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Overview

Current methods of water-borne pathogen detection require expensive equipment that often isn't modular or accessible to developing countries. Thus, current sensors are limited to specific bacterial strains or are too broad and produce false positive tests. 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 that each contain 23 amino acids 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.

Functionally, this interaction allowed us to take 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/]} when binding to the membranes of bacteria, the close proximity of the linked split beta-galactosidase units can be achieved. Previous research indicates that this reporter system can be applied to mammalian cell detection with different ligands in close proximity. The novel approach of this project involves linking Magainin to the beta-galactosidase reporter system to detect pathogenic bacteria. The benefit of this system is its modularity. The magainin peptide can be switched out with other antimicrobial peptides - which may have better binding affinities to other bacterial strains - via BioBrick assembly, creating a system that can be engineered to potentially detect any bacterial strain.

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.