Difference between revisions of "Team:Exeter/Design"

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Revision as of 14:49, 25 October 2017

Type I Pili

Pili are small, hair-like protein structures on the surface of bacterial cells, that are used for cell-cell signalling and biofilm formation. Their most significant ability is mannose-binding. This is integral to the mechanism of the infection of the human urinary tract by E. coli . We have focused on type I pili, which are present in some gram-negative bacteria such as E. coli and coded for by the fim operon. The type I pili are a complex of a number of Fim proteins, some of which make up the structural body of the projection, others are integral to the the chaperone-usher pathway, while the terminal FimH protein is responsible for binding to mannose.

Plasmid Design

We have produced two types of plasmids, using the modular cloning method *reference* 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 *insert reference* at the 22nd amino acid residue. Based on the primary water samples 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 and 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

The genetically modified, metal ion extracting bacteria will be used in a three-stage filter system, consisting of the hydrocyclone, fluidised media reactor and a biosecurity mechanism. The GM bacteria will be housed in the fluidised media reactor, where the pili will be able to adsorb 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)