Team:British Columbia/Project DspB

From 2010.igem.org

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<h3>Introduction</h3>
<h3>Introduction</h3>
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<p>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 <i>Escherichia coli</i> (<i>E. coli</i>)and <i>Staphylococcus epidermidis</i>. 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 <i>Actinobacillus actinomycetemcomitans</i> and utilize it in concurrence with a phage to effectively eliminate <i>Staphylococcus aureus</i> biofilms. </p>
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<p>Dispersin B (DspB) is an enzyme that degrades biofilms by catalyzing the hydrolysis of poly-ß-(1,6)-linked N-acetylglucosamine bonds. These bonds exist in the extracellular polymeric substance (EPS), which acts as a polysaccharide adhesin relevant to biofilm formation and integrity in <i>Escherichia coli</i> and <i>Staphylococcus epidermidis</i>. According to Lu and Collins (reference), DspB effectively cleaves these bonds and impedes biofilm formation. The DspB sub-team's goal was to isolate DspB from its natural host <i>Actinobacillus actinomycetemcomitans</i> and incorporate it into a phage to effectively eliminate <i>Staphylococcus aureus</i> biofilms.</p>
<h3>Approach</h3>
<h3>Approach</h3>
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<p>We ordered  <i>Actinobacillus actinomycetemcomitans</i> strain HK1651 from <a href="http://www.atcc.org/">ATCC</a> and proceeded to obtain the genetic code of the protein. We designed two sets of primers to PCR the sequence off the genome: <br>
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<p>We ordered  <i>Actinobacillus actinomycetemcomitans</i> strain HK1651 genomic DNA from <a href="http://www.atcc.org/">ATCC</a> and designed two sets of primers to PCR the sequence for DspB off the genome: <br>
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1) Primers containing a histidine tag<br>
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Set (1) Primers with a histidine tag<br>
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2) Primers that <b>do not</b> contain the histidine tag<br></p>
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Set (2) Primers without a histidine tag<br></p>
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<p>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 (psb1c3). </p>
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<p>The PCR with set (2) primers was successful and we created a standard biobrick part with the appropriate flanking restriction sites, EcoRI, XbaI, SpeI, PstI, on the required chloramphenicol resistant backbone (psb1c3). </p><br/>
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<p> In order to express the protein in <i>E. coli</i>, we need to build the following construct:
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<p>In order to express the DspB protein in <i>E. coli</i>, we aimed to build the following construct:
<center><src><img src="https://static.igem.org/mediawiki/2010/3/31/Dspb_construct.png" width=200px></src></center></br>
<center><src><img src="https://static.igem.org/mediawiki/2010/3/31/Dspb_construct.png" width=200px></src></center></br>
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<p><caption>Figure 1. dspB construct with a constitutive promoter, ribosome binding site, dspB, and terminator</caption></p><br>
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<p><caption>Figure 1. Construct consisting of a constitutive promoter, ribosome binding site, gene encoding DspB, and terminator.</caption></p><br/>
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<p>We used the constitutive promoter (insert part number) with a ribosome binding site (insert part number)and relied on the internal stop codon native to <i>dspB</i>. After expressing this protein in <i>E. coli</i>, we want to isolate DspB using the histidine tag (a more specific approach) as well as lysing the cell to obtain the crude lysate (a more general approach). Due to unforeseen circumstances, we could only perform the latter. </p>
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<p>We used the constitutive promoter (insert part number) with a ribosome binding site (insert part number)and relied on the terminator already present in the psb1c3 plasmid. The original plan was to isolate DspB using the histidine tag as well as lyse the cell to obtain crude lysate. However, since only the PCR with set (2) primers worked, we continued our experiments using crude lysate.</p>
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<p>To characterize DspB's ability to degrade poly-ß-(1,6)-linked N-acetylglucosamine bonds, we performed SUBSTRATE assays.</P><BR/>
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<p>To test whether or not DspB will actually degrade poly-ß-(1,6)-linked N-acetylglucosamine bonds, we ordered said substrate and conducted crude cell assay experiments (The results are below).
 
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<p>The next steps are to test dspB on a <i>S. aureus</i> biofilm, encode dspB into the phage, and together, with the phage, be exposed to a <i>S. aureus</i> biofilm.
 
<h3>Results & Discussion</h3>
<h3>Results & Discussion</h3>
<center><img src="https://static.igem.org/mediawiki/2010/c/c6/DspB_Assay_2.jpg" width=600px></center>
<center><img src="https://static.igem.org/mediawiki/2010/c/c6/DspB_Assay_2.jpg" width=600px></center>
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<p>Future directions include testing DspB's effect on a <i>S. aureus</i> biofilm, as well as incorporating DspB into the phage for exposure to <i>S. aureus</i> biofilms.</p>

Revision as of 16:02, 26 October 2010



Introduction

Dispersin B (DspB) is an enzyme that degrades biofilms by catalyzing the hydrolysis of poly-ß-(1,6)-linked N-acetylglucosamine bonds. These bonds exist in the extracellular polymeric substance (EPS), which acts as a polysaccharide adhesin relevant to biofilm formation and integrity in Escherichia coli and Staphylococcus epidermidis. According to Lu and Collins (reference), DspB effectively cleaves these bonds and impedes biofilm formation. The DspB sub-team's goal was to isolate DspB from its natural host Actinobacillus actinomycetemcomitans and incorporate it into a phage to effectively eliminate Staphylococcus aureus biofilms.

Approach

We ordered Actinobacillus actinomycetemcomitans strain HK1651 genomic DNA from ATCC and designed two sets of primers to PCR the sequence for DspB off the genome:
Set (1) Primers with a histidine tag
Set (2) Primers without a histidine tag

The PCR with set (2) primers was successful and we created a standard biobrick part with the appropriate flanking restriction sites, EcoRI, XbaI, SpeI, PstI, on the required chloramphenicol resistant backbone (psb1c3).


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


Figure 1. Construct consisting of a constitutive promoter, ribosome binding site, gene encoding DspB, and terminator.


We used the constitutive promoter (insert part number) with a ribosome binding site (insert part number)and relied on the terminator already present in the psb1c3 plasmid. The original plan was to isolate DspB using the histidine tag as well as lyse the cell to obtain crude lysate. However, since only the PCR with set (2) primers worked, we continued our experiments using crude lysate.

To characterize DspB's ability to degrade poly-ß-(1,6)-linked N-acetylglucosamine bonds, we performed SUBSTRATE assays.


Results & Discussion

Future directions include testing DspB's effect on a S. aureus biofilm, as well as incorporating DspB into the phage for exposure to S. aureus biofilms.


References

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