Team:British Columbia/Project DspB

Introduction

Dispersin B (dspB) is an enzyme that degrades biofilms by catalyzing the hydrolysis of poly-ß-(1,6)-linked N-acetylglucosamine bond. These bonds exist as a polymer in the extracellular polymeric substance (EPS) as a polysaccharide adhesin; this adhesin is relevant in biofilm formation and integrity in both Escherichia coli (E. coli)and Staphylococcus epidermidis. According to Lu and Collins (reference paper), dspB effectively cleaves these bonds, thus affecting biofilm formation. Our goal is to isolate dspB from the host Actinobacillus actinomycetemcomitans and utilize it in concurrence with a phage to effectively eliminate Staphylococcus aureus biofilms.

Approach

We ordered Actinobacillus actinomycetemcomitans strain HK1651 from ATCC and proceeded to obtain the genetic code of the protein. We created and used two sets of primers to PCR the sequence off the genome:
1) Primers containing 6 histidines
2) Primers that do not contain histidines

After attaining the sequence, we created a standard biobrick part with the appropriate flanking restriction sites, EcoRI, XbaI, SpeI, PstI, on a chloramphenicol resistant backbone, namely psb1c3.

In order to express the protein in E. coli, we need to build the following construct:

Figure 1. dspB construct with a constitutive promoter, ribosome binding site, dspB, and terminator

After expressing this protein in E. coli, we want to purify dspB using the histidine tag (a more specific approach) as well as isolate the protein by lysing the cell to obtain the crude lysate (a more general approach). Due to unforeseen circumstances, we could only perform the latter.

To test whether or not dspB will actually degrade the poly-ß-(1,6)-linked N-acetylglucosamine bonds, we ordered said substrate and conducted crude cell assay experiments (The results are below).

The next steps are to test dspB on a S. aureus biofilm, encode dspB into the phage, and together, with the phage, be exposed to a S. aureus biofilm.

Results

Figure 2. dspB crude cell assay depicting the activity of the enzyme (dspB) on the substrate (signified by NP) poly-ß-(1,6)-linked N-acetylglucosamine