Team:Oxford/Parts

Parts

Introduction


The aim of our project is to engineer bacteria into living therapeutics that can be used to treat biofilm-associated urinary tract infections (UTIs). To that end, we have created a series of BioBricks that code for antibiofilm and antibacterial proteins such that we can investigate their function and evaluate their suitability for use as a UTI therapeutic.
* Note: The relevant literature references for each part, as well as their full characterization data (where applicable) can be found at their respective pages in the Registry of Standard Biological Parts.
** Note: Unless otherwise stated, all our parts containing protein-coding sequences contain a hexahistidine tag-coding sequence fused downstream of our gene of interest to facilitate protein detection and purification.


Biofilm-degrading proteins


We have identified the exopolysaccharides and extracellular DNA produced by bacteria as major structural components of bacterial biofilms which can serve as targets for biofilm degradation. As such, we obtained nucleotide sequences for enzymes known to target these components and catalogued them as BioBricks after making appropriate modifications to suit our needs:


Dispersin B

Dispersin B (DspB) is an enzyme that specifically hydrolyses the β-1,6-glycosidic linkages found in the exopolysaccharides of ''E. coli'' and ''S. aureus'' biofilms, among others, ''E. coli'' being the primary pathogen that causes UTIs.

BBa_K1659200

This basic part contains the gene coding for DspB.

BBa_K1659210

This basic part contains the gene coding for DspB with a single point mutation at nucleotide 582. The mutation was introduced to remove the BspHI restriction site within the sequence, as we need the sequence to be free of BspHI sites when we insert the gene into the commercial expression vector pBAD/HisB for part characterization.

BBa_K1659201

To facilitate the export of DspB from its expression host cell, we created this composite part in which the coding sequence for the DsbA 2-19 secretion tag was fused upstream of the coding sequence for DspB.

BBa_K1659211

Similarly to part BBa_K1659210, a point mutation was introduced into the sequence of DsbA-DspB to remove an offending BspHI restriction site that posed a hindrance to us inserting the gene into the commercial expression vector pBAD/HisB for part characterization.

Micrococcal DNase

Micrococcal DNase is an enzyme that is capable of hydrolysing the extracellular DNA found in bacterial biofilms:

BBa_K1659300

This part contains the coding sequence for the protein Micrococcal DNase with a DsbA 2-19 secretion tag fused to its N-terminus.