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Team:British Columbia/Project DspB
From 2010.igem.org
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<h3>Introduction</h3> | <h3>Introduction</h3> | ||
| - | <p>Dispersin B ( | + | <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> |
<h3>Approach</h3> | <h3>Approach</h3> | ||
<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> | <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> | ||
| - | 1) Primers containing | + | 1) Primers containing a histidine tag<br> |
| - | 2) Primers that <b>do not</b> contain | + | 2) Primers that <b>do not</b> contain the histidine tag<br></p> |
| - | <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 | + | <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> |
<p> In order to express the protein in <i>E. coli</i>, we need to build the following construct: | <p> In order to express the protein in <i>E. coli</i>, we need 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> | ||
<p><caption>Figure 1. dspB construct with a constitutive promoter, ribosome binding site, dspB, and terminator</caption></p><br> | <p><caption>Figure 1. dspB construct with a constitutive promoter, ribosome binding site, dspB, and terminator</caption></p><br> | ||
| + | <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> | ||
| - | + | <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). | |
| - | + | ||
| - | <p>To test whether or not | + | |
<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. | <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> | ||
Revision as of 09:18, 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 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 designed two sets of primers to PCR the sequence off the genome:
1) Primers containing a histidine tag
2) Primers that do not contain the histidine tag
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).
In order to express the protein in E. coli, we need to build the following construct:
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 dspB. After expressing this protein in E. coli, 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.
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).
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 & Discussion
