Biological Design

Type I Pili

Pili are small, hair-like structures found on bacterial cells. Pili are encoded by the fim operon which contains a number of genes that code for structural proteins, including FimH. The main constituent is FimA which polymerises to form the main body of the pilus structure and the other proteins are largely related to the chaperone-usher pathway (Busch et. al 2015). FimH is found at the very end of the complex, and contains lectin - the mannose binding domain. They have a natural ability to attach to eukaryotic cell surface mannose molecules due to specific binding sites within FimH, the terminal protein. By investigating adhesion mechanisms involved in type I pili we aimed to repurpose the structures by introducing new metal-binding domains within the pili of our E. coli.

We have produced two types of plasmid, using the modular cloning method (Weber, E., et al) that, once transformed into a bacterium, will be able to synthesise the engineered pili capable of binding to metal ions in water. To ascertain the ideal placement of the metal-binding coding sequences, we tested the expression of super-folded GFP at the 22nd, 225th and 258th amino acid. Protein-coding sequences have previously been inserted at these three positions, by numerous research groups. Based on our research and results, we decided to insert all the metal-binding proteins at the 22nd amino acid.

The first plasmid contains the fimH gene, with an inserted metal-binding protein immediately at the end of the signal peptide at the 22nd amino acid residue. This is intended to minimise issues with the chaperone-usher pathway and open up further opportunities for future FimH modification. Based on the primary water samples we collected from the abandoned Wheal Maid mine, we have chosen to insert four different metal-binding proteins that bind the most present and damaging ions. We have decided to make four different plasmids, with four different metal-binding proteins:

The second plasmid contains the remainder of the fim operon containing five fim coding sequences excluding fimH to allow the biosynthesis of the entire pilus structure. This plasmid always needs to be co-transformed with the fimH plasmid that codes for the pertinent fusion protein to produce metal binding pili. This can be used as a reproducible method for modifying the fim operon in pili by producing a new modular toolkit to advance the field of metal extraction.

Real Life Application

We are aware of the high risks associated with involving a genetically modified organism within a real life application. As demonstrated in the research conducted by the Exeter 2016 iGEM team a kill switch is not a reliable biocontainment method. To take this into account we have developed three-stage filteration system, consisting of the hydrocyclone, metal binding reactor and a biosecurity mechanism. The GM bacteria will be contained within the metal binding reactor, where the pili will be able to sequester metal ions from water. The water then passes into the biosecurity mechanism, which will kill any bacteria that try to escape.

References

• Fronzes, R. et al., Architectures and biogenesis of non‐flagellar protein appendages in Gram‐negative bacteria, The EMBO Journal, Volume 27, Issue 17, 2271-2352 (2008)
• Pallesen, L. et al., Chimeric FimH adhesin of type 1 fimbriae: a bacterial surface display system for heterologous sequences, Microbiology, 141, 2839-2848 (1995)
• Weber, E., Engler, C., Gruetzner, R., Werner, S., and Marrillonnet, S. (2011) A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS One 6, e16765.