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), as a novel alternative to conventional small-molecule antibiotics. 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.
We submitted 12 parts to the Registry, all of which were sequence-confirmed. Our parts are presented in table view at our Registry summary page.
* 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 Escherichia coli and Staphylococcus aureus biofilms, among others, E. coli being the primary pathogen that causes UTIs.
BBa_K1659200: DspB
This basic part contains the gene coding for DspB.
BBa_K1659210: DspBx
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: DsbA-DspB
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 DsbA-DspBx
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_K1659301: DsbA-DNase
This part contains the coding sequence for the protein Micrococcal DNase with a DsbA 2-19 secretion tag fused to its N-terminus.
Bacteria-killing Proteins
Biofilm-degrading proteins alone cannot fully treat UTIs as they only destroy the biofilm matrix encasing the bacterial cells but not the cells themselves. As such, we have also designed BioBrick parts containing genes encoding for proteins that directly kill bacteria:
Artilysin Art-175
Art-175 is a fusion protein that kills Gram-negative bacteria, including major UTI-causing pathogens such as E. coli and Pseudomonas aeruginosa by means of bypassing their outer membranes and catalysing the hydrolysis their cell walls.
BBa_K1659000: Art175
This part contains the gene coding for Art-175.
BBa_K1659001: Fla-Art175
To facilitate the export of Art-175 from its expression host cell, we created this composite part in which the coding sequence for the flagellin 26-47 secretion tag was fused upstream of the coding sequence for Art-175.
BBa_K1659002: DsbA-Art175
To facilitate the export of Art-175 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 Art-175.
BBa_K1659003: YebF-Art175
To facilitate the export of Art-175 from its expression host cell, we created this composite part in which the coding sequence for the YebF secretion tag was fused upstream of the coding sequence for Art-175.
Microcin S
Microcin S (MccS) is a narrow-spectrum antibacterial protein that has been shown to exhibit high-potency killing of select strains of E. coli and P. aeruginosa. A narrow-spectrum antibacterial is especially useful in our medical context because in a real-world application we will want to kill pathogenic bacteria in a specific manner while causing as minimal a degree of harm to the wider microbiota of the urinary tract as possible (see our brief urinary tract microbiome survey).
BBa_K1659100: MccS
This part contains the gene coding for MccS.
Art-E
We created the coding sequence for our own novel fusion protein Art-E, by fusing the gene for membrane disruption protein SMAP-29 upsteam of the gene for cell wall lysis protein T4 lysozyme. We theorize that the resulting protein, being the combination of two broad-spectrum antimicrobials, should exhibit broad spectrum antibacterial activity.
BBa_K1659088: ArtE
This part contains the coding sequence for Art-E.
Quorum Sensing
A possible extension to our project is the incorporation of a quorum sensing element to our antibacterial and antibiofilm secretion designs. Linking the gene expression for our secreted biofilm-degrading and bacteria-killing proteins to a promoter which is activated by the quorum sensing signals used by the target pathogen would allow the creation of a responsive pathogen killer. In view of that, we created a part which expresses the gene coding for a reporter protein in response to E. coli quorum sensing signals.
pLsr-RBS-GFP
pLsr is a promoter which is induced by E. coli AI-2 quorum sensing signals via an indirect mechanism. By linking a GFP reporter gene downstream of pLsr, this part design allows us to test the promoter's responsiveness to AI-2.