Difference between revisions of "Team:Exeter/Design"

Project Design

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Pili are small, hair-like protein structures on the surface of bacterial cells, that are used for cell-cell signalling and biofilm formation. 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 consist of Fim proteins and are synthesised using the chaperone-usher pathway.

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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 test the most efficient place to insert 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 protein, with an inserted metal-binding protein at the 22nd amino acid to allow metal ion binding. Based on the primary water samples collected from the abandoned Wheal Maid mine, we have chosen to insert four different metal-binding proteins that were present in the water samples. 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 proteins and excluding FimH to allow the biosynthesis of the entire pilus structure. This plasmid always needs to be co-transformed with the desired FimH_22_Fusion Protein to produce genetically engineered 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.

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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)
• Miller, E. et al., The Mechanical Properties of E.coli Type 1 Pili Measured by Atomic Force Microscopy Techniques, Biophysical Journal, Volume 91, 3848-3856 (2006)
• Munera, D. et al., Recognitiion of the N-terminal lectin domain of FimH adhesin by the usher FimD is required for type 1 pilus biogenesis, Molecular Biology, 64(2), 333-346 (2007)
• Pallesen, L. et al., Chimeric FimH adhesin of type 1 fimbriae: a bacterial surface display system for heterologous sequences, Microbiology, Volume 141, 2839-2848 (1995)