Difference between revisions of "Team:Lethbridge HS/Results"

 
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                     <a href="https://2015.igem.org/Team:Lethbridge_HS/Introduction" id="nav-head" class="navbar-brand" style="margin-top:10px;"><h1 id="headerText" style="font-weight:100; font-family: 'Mohave'; ">Lethbridge iGEM      </h1></a>
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                     <a href="https://2015.igem.org/Team:Lethbridge_HS/Introduction" id="nav-head" class="navbar-brand" style="margin-top:10px;"><h1 id="headerText" style="font-weight:100; font-family: 'existence'; ">Lethbridge iGEM      </h1></a>
 
                  
 
                  
  
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                                     <a href="https://2015.igem.org/Team:Lethbridge_HS/Description">Description</a>
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                                     <a href="https://2015.igem.org/Team:Lethbridge_HS/Description">Background</a>
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                                     <a href="#">Parts</a>
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<a href="https://2015.igem.org/Team:Lethbridge_HS/Experiments">Experiments</a>
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<a href="#">Achievements</a>
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<a href="https://2015.igem.org/Team:Lethbridge_HS/Results">Results</a>
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                                     <a href="https://2015.igem.org/Team:Lethbridge_HS/Parts">Parts</a>
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<a href="https://2015.igem.org/Team:Lethbridge_HS/Achievements">Achievements</a>
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                         <li margin-top:2%; style="margin-top:2%;"><a class="textItem" href="https://2015.igem.org/Team:Lethbridge_HS/Notebook">Notebook</a></li>
 
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                                     <a href="https://2015.igem.org/Team:Lethbridge_HS/Team">Students</a>
 
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                                     <a href="#">Advisors</a>
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                                     <a href="https://2015.igem.org/Team:Lethbridge_HS/Team#section2">Advisors</a>
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<a class="texItem" href="#">Sponsors</a>
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<a class="texItem" href="https://2015.igem.org/Team:Lethbridge_HS/Team#section3">Sponsors</a>
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<a class="texItem" href="#">Attributions</a>
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<a class="texItem" href="https://2015.igem.org/Team:Lethbridge_HS/Attributions">Attributions</a>
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<a class="texItem" href="#">Collaborations</a>
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<a class="texItem" href="https://2015.igem.org/Team:Lethbridge_HS/Collaborations">Collaborations</a>
 
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                 <p id="pageTitleText">Project<br></p><p id="pageSubtitleText"><span>How does our project work?</span></p>
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                 <p id="pageTitleText">Results<br></p><p id="pageSubtitleText"><span>What results did we get in the lab?</span></p>
 
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                             <section id="section1">
 
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<h1 id="projecttext1" class="contentSubTitle"> Description <br><small></small></h1>
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</section>
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<p id="humanpractices_hp" class="bees">Without the bees planning dinner would be significantly more difficult. Bees pollinate 70 out of the top 100 food crops, which supply 90% of the world nutrition. It is apparent that bees are an integral part of the ecosystem and human life. However, bees have been in decline for about 30 years, but the rate of deaths have gone up in the past decade. In the United States, a startling 30% of bees are dying each year and this is due to a phenomenon called Colony Collapse Disorder (CCD) which is destroying productive bee colonies worldwide. One factor contributing to CCD is the parasitic mite, Varroa destructor. The parasite sucks the bees’ haemolymph (blood), and transmits RNA viruses, such as Deformed Winged Virus, which are detrimental to colony productivity. Current methods used to control V. destructor are inefficient and resistance is developing in treated populations. Using synthetic biology, we designed E.coli that produce the miticide oxalic acid in the bee gut. This method targets V. destructor by directly delivering oxalic acid into the mites, creating mite-proof bee populations. </p>
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<h1 id="projecttext1" class="contentSubTitle"> Results <br><small></small></h1>
  
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<img src="https://static.igem.org/mediawiki/2015/5/55/LethHS2015_1234.jpg" width=300px height=150px class="img-responsive>
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<p id="humanpractices_hp" class="bees"><b>Oxalic acid results:</b><br></p>
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<p>efrggnbtnhekrtbwekjgt efrggnbtnhekrtbwekjgtbefrggnbtnhekrtbwekjgtbefrggnbtnhekrtbwekjgtbefrggnbtnhekrtbwekjgtbefrggnbtnhekrtbwekjgtbefrggnbtnhekrtbwekjgtbefrggnbtnhekrtbwekjgtbefrggnbtnhekrtbwekjgtbefrggnbtnhekrtbwekjgtbefrggnbtnhekrtbwekjgtbefrggnbtnhekrtbwekjgtbefrggnbtnhekrtbwekjgtbefrggnbtnhekrtbwekjgtbefrggnbtnhekrtbwekjgtbefrggnbtnhekrtbwekjgtbefrggnbtnhekrtbwekjgtbb</p>
+
  
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<p id="humanpractices_hp" class="biofilms">The purpose of hospitals is to help people get better. However, in the United States, 2 million people are infected during their hospital stay and bacterial biofilms are responsible of 65% of all hospital acquired infections. A biofilm is a conglomeration of bacteria that is enclosed in a matrix of sugars and extracellular DNA, this helps to hold the bacteria together like a community. Biofilms can adhere to any surface and are commonly found in nature. However, biofilms can become problematic when they adhere to surgical tools such as catheters, endotracheal tubes, and scalpels. Currents methods used to destroy biofilms include antibiotics and biocides. These methods are often expensive, harsh, and ineffective; biofilms are notorious for developing resistance to chemical treatments. Instead of trying to kill the bacteria in the biofilm, we decided to degrade the matrix that protects it. We have created a cocktail of Nuclease and Dextranase to achieve this purpose. Once the matrix is degraded, the bacteria inside can be eliminated without the use of expensive chemicals.<br><br>
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<div style="width:60%;" class="bees">
 +
<table class="table table-striped table-bordered">
 +
                                            <thead>
 +
                                                <tr>
 +
                                                    <th>Oxalic Acid Concentration (mg/mL)</th>
 +
                                                    <th>OD528</th>
 +
                                                </tr>
 +
                                            </thead>
 +
                                            <tbody>
 +
                                                <tr>
 +
                                                    <td>0.1</td>
 +
                                                    <td>1.862</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>0.2</td>
 +
                                                    <td>1.675</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>0.3</td>
 +
                                                    <td>1.675</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>0.4</td>
 +
                                                    <td>1.655</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>0.5</td>
 +
                                                    <td>1.673</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>0.6</td>
 +
                                                    <td>1.680</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>0.7</td>
 +
                                                    <td>1.655</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>0.8</td>
 +
                                                    <td>1.630</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>0.9</td>
 +
                                                    <td>1.587</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>1.0</td>
 +
                                                    <td>1.564</td>
 +
                                                </tr>
  
−
<b>What is nuclease? What is dextranase?</b><br>
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                                            </tbody>
−
As mentioned earlier, one of the two targets for dispersing the biofilm is its extracellular DNA. To do so, we are using E.coli to secrete nuclease. A nuclease is an enzyme that catalyzes the hydrolysis of the phosphodiester bonds of nucleic acids. More specifically, the nuclease utilized by our team is a Micrococcal nuclease (Mnase) capable of cleaving single-stranded and double-stranded nucleic acids. The irony of the situation being that the nuclease bio brick (Part: BBa_K729004) being used is derived from Staphylococcus aureus.  S. aureus biofilms are a major problem on medical equipment, and account for many infections. The S. aureus biofilm uses the Mnase for partial dispersal of its outer layer, so its inner colonies can spread outside and grow more biofilms. S aureus. Being a gram positive, ubiquitous bacteria, has one of the toughest biofilms. And hence, the strength of the Mnase is equally supplementary. Although the Mnase is derived from S. aureus, the function and the effect of the enzyme is not limited to that bacteria. Since extracellular DNA is component of almost every biofilm, the effect of the enzyme will not be affected by a change in the species/strain of bacteria. To further enhance the effects of Mnase, it will be applied in a mixture that also includes Dextranase. Dextranase is an enzyme that catalyzes the hydrolysis of bonds within dextran. Dextran is a component of the exopolysaccharide (EPS) matrix, common to many biofilms. The dextranase used for the construct, is an alpha-dextranase derived from Chaetomium gracile (a dematiaceous mold from the fungi family). Dextranase, by degrading parts of the EPS matrix would allow for Mnase to further seep into the biofilm, and thus increase the overall efficiency of the mixture<br><br>
+
                                        </table>
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+
</div>
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<b>What we are doing differently</b><br>
+
−
Past projects have targeted the bonds within the biofilm structure to disperse the biofilms, but since there is a variety of major bonds found within different biofilms, the effects of the construct have been limited to some bacterial species. But, by targeting the extracellular DNA and the exopolysaccharide matrix (common components of almost every biofilm), our aim is to create a general all-purpose dispersant, capable of working on a variety of biofilms, thriving in a variety of settings.<br><br>
+
  
−
<b>Extracellular Polymeric Substance Matrix: Sticky Stuff!</b><br>
 
−
The bacteria in the biofilm are surrounded by an extracellular polymeric substance (EPS) matrix. This matrix is comprised of water, which hydrates the cells; various sugars, to provide nutrients and the sticky structure of the biofilm; proteins, which are typically enzymes; lipids; and extracellular DNA, which serves as a structural component of the biofilms. The EPS matrix constitutes 50-90% of a biofilm’s organic matter. The purpose of the EPS matrix is to adhere the bacteria to a surface and protect the biofilm against any harsh environmental conditions. The matrix also provides a “transport system” so that nutrients, water, and enzymes can move around the structure to meet the needs of each cell.<br><br>
 
  
−
The production of the EPS matrix is, in part, regulated by quorum sensing; this is a way for bacteria to communicate with each other via chemical signalling molecules. Like a “quorum”, once there are enough bacteria, the bacteria are able to communicate with each other to collectively express a gene, in this is case they would be producing the EPS.
 
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</p>
 
  
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</section>
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<section id="section2">
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<img src="https://static.igem.org/mediawiki/2015/c/c9/LethHS2015_oxalicacidcurve.jpg" style="" width="600px" height="400px" class="bees img-responsive">
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</section>
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 +
<p id="humanpractices_hp" class="bees"><b>Bee trials:</b><br></p>
 +
 
 +
 
 +
<table class="table table-striped table-bordered bees"> <caption></caption>
 +
                                            <thead>
 +
                                                <tr>
 +
                                                    <th>Treatment</th>
 +
                                                    <th>Before</th>
 +
<th>After</th>
 +
                                                </tr>
 +
                                            </thead>
 +
                                            <tbody>
 +
                                                <tr>
 +
                                                    <td>None</td>
 +
                                                    <td>Somewhat lethargic. Drank when prompted.</td>
 +
<td>Appeared tired, but otherwise unaffected.</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>0.00%</td>
 +
                                                    <td>Most lethargic, did not eat when offered</td>
 +
<td>Wet and slow moving.</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>0.05%</td>
 +
                                                    <td>Very vigorous; ran around inside the tube. Fed.</td>
 +
<td>Very energetic. Unaffected by treatment.</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>0.2%</td>
 +
                                                    <td>Very vigorous; ran around inside the tube. Fed.</td>
 +
<td>Slightly less energetic than the 0.05% bee.</td>
 +
                                                </tr>
 +
                                             
 +
 
 +
                                                   
 +
                                            </tbody>
 +
                                        </table>
 +
<p class="bees">The bees will be able to survive all of the oxalic acid that the E.coli can produce.</p>
 +
<p id="humanpractices_hp" class="bees"><b>Mite trials:</b><br></p>
 +
<table class="table table-striped table-bordered bees"> <caption></caption>
 +
                                            <thead>
 +
                                                <tr>
 +
                                                    <th></th>
 +
                                                    <th>Control</th>
 +
<th>Oxalic Acid</th>
 +
<th>RFP</th>
 +
                                                </tr>
 +
                                            </thead>
 +
                                            <tbody>
 +
                                                <tr>
 +
                                                    <td><b>Parafilm Pouch</b></td>
 +
                                                    <td>Mites dead</td>
 +
<td>Mites dead</td>
 +
<td>Mites dead</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                                                                        <td><b>Larvae 1</b></td>
 +
                                                    <td>Mites dead</td>
 +
<td>Mites dead</td>
 +
<td>Mites dead</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                                                                                                                        <td><b>Larvae 2</b></td>
 +
                                                    <td>Mites dead</td>
 +
<td>Mites dead</td>
 +
<td>Mites dead</td>
 +
                                                </tr>
 +
                                                           
 +
                                            </tbody>
 +
                                        </table>
 +
<p class="bees">The mites did not survive any of the trials.</p>
 +
 
 +
 
 +
<p id="humanpractices_hp" class="biofilms"><b>Antibiotics testing:</b><br></p>
 +
<table class="table table-striped table-bordered biofilms"> <caption></caption>
 +
                                            <thead>
 +
                                                <tr>
 +
                                                    <th>Test (Antibiotics)</th>
 +
                                                    <th>Result</th>
 +
<th>After</th>
 +
                                                </tr>
 +
                                            </thead>
 +
                                            <tbody>
 +
                                                <tr>
 +
                                                    <td>AMP 1</td>
 +
                                                    <td>Positive</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>AMP 2</td>
 +
                                                    <td>Positive</td>
 +
<td>Wet and slow moving.</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>CAM 1</td>
 +
                                                    <td>Positive</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>CAM 2</td>
 +
                                                    <td>Positive</td>
 +
                                                </tr>
 +
  <tr>
 +
                                                    <td>TET 1</td>
 +
                                                    <td>Positive</td>
 +
                                                </tr>  <tr>
 +
                                                    <td>TET 2</td>
 +
                                                    <td>Positive</td>
 +
                                                </tr>
 +
<tr>
 +
                                                    <td>Control (water)</td>
 +
                                                    <td>Positive</td>
 +
                                                </tr>
 +
                                             
 +
 
 +
                                                   
 +
                                            </tbody>
 +
                                        </table>
 +
<p class="biofilms">The results we got for antibiotics testing show that biofilms can still be recultured even after being treated with antibiotics.</p>
 +
 
 +
<p class="biofilms"><b>Testing with cleaners</b></p>
 +
<table class="table table-striped table-bordered biofilms"> <caption></caption>
 +
                                            <thead>
 +
                                                <tr>
 +
                                                    <th>Cleaner</th>
 +
                                                    <th>Results</th>
 +
<th>After</th>
 +
                                                </tr>
 +
                                            </thead>
 +
                                            <tbody>
 +
                                                <tr>
 +
                                                    <td>Bleach</td>
 +
                                                    <td>-</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>Tilex</td>
 +
                                                    <td>3</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>Mr. Clean</td>
 +
                                                    <td>-</td>
 +
                                                </tr>
 +
                                                <tr>
 +
                                                    <td>Fantastik</td>
 +
                                                    <td>1</td>
 +
                                                </tr>
 +
  <tr>
 +
                                                    <td>Truly</td>
 +
                                                    <td>2</td>
 +
                                                </tr>
 +
  <tr>
 +
                                                    <td>Windex</td>
 +
                                                    <td>1</td>
 +
                                                </tr>
 +
  <tr>
 +
                                                    <td>Lysol</td>
 +
                                                    <td>-</td>
 +
                                                </tr>
 +
  <tr>
 +
                                                    <td>GreenWorks</td>
 +
                                                    <td>1</td>
 +
                                                </tr>
 +
                                             
 +
 
 +
                                                   
 +
                                            </tbody>
 +
                                        </table>
 +
 
 +
 
 +
 
 +
 
 +
<figure style=" margin-right:20px; float:left;" class="biofilms">
 +
<img src="https://static.igem.org/mediawiki/2015/a/af/LethHS2015_5.png" width=400px height=300px class="img-responsive">
 +
<figcaption>Biofilms fluorescent imaging under Typhoon</figcaption>
 +
</figure>
 +
 
 +
<figure style=" margin-right:20px; float:left;" class="bees">
 +
<img src="https://static.igem.org/mediawiki/2015/1/1c/LethHS2015_6.jpg" width=400px height=300px class="img-responsive">
 +
<figcaption>Different concentration of oxalic acid 3 minutes into incubation time at RT</figcaption>
 +
</figure>
 +
 
 +
<figure style=" margin-right:20px; float:left;" class="bees">
 +
<img src="https://static.igem.org/mediawiki/2015/4/48/LethHS2015_7.png" width=400px height=300px class="img-responsive">
 +
<figcaption>E. coli cultures under varying concentration of oxalic acid</figcaption>
 +
</figure>
 +
 
 +
<figure style=" margin-right:20px; float:left;" class="bees">
 +
<img src="https://static.igem.org/mediawiki/2015/c/c3/LethHS2015_8.jpg" width=400px height=300px class="img-responsive">
 +
<figcaption>E. coli cultures under varying concentration of oxalic acid</figcaption>
 +
</figure>
 +
 
 +
<figure style=" margin-right:20px; float:left;" class="bees">
 +
<img src="https://static.igem.org/mediawiki/2015/f/fd/LethHS201510.png" width=400px height=300px class="img-responsive">
 +
<figcaption>Microcentrifuge tube containing Honey Bee</figcaption>
 +
</figure>
 +
 
 +
<figure style=" margin-right:20px; float:left;" class="bees">
 +
<img src="https://static.igem.org/mediawiki/2015/d/d3/LethHS201511.png" width=400px height=300px class="img-responsive">
 +
<figcaption>Microcentrifuge tube containing Varroa mites</figcaption>
 +
</figure>
 +
 
 +
 
  
 
                     </div>
 
                     </div>

Latest revision as of 03:46, 19 September 2015

Results

What results did we get in the lab?

Results

Oxalic acid results:

Oxalic Acid Concentration (mg/mL) OD528
0.1 1.862
0.2 1.675
0.3 1.675
0.4 1.655
0.5 1.673
0.6 1.680
0.7 1.655
0.8 1.630
0.9 1.587
1.0 1.564

Bee trials:

Treatment Before After
None Somewhat lethargic. Drank when prompted. Appeared tired, but otherwise unaffected.
0.00% Most lethargic, did not eat when offered Wet and slow moving.
0.05% Very vigorous; ran around inside the tube. Fed. Very energetic. Unaffected by treatment.
0.2% Very vigorous; ran around inside the tube. Fed. Slightly less energetic than the 0.05% bee.

The bees will be able to survive all of the oxalic acid that the E.coli can produce.

Mite trials:

Control Oxalic Acid RFP
Parafilm Pouch Mites dead Mites dead Mites dead
Larvae 1 Mites dead Mites dead Mites dead
Larvae 2 Mites dead Mites dead Mites dead

The mites did not survive any of the trials.

Antibiotics testing:

Test (Antibiotics) Result After
AMP 1 Positive
AMP 2 Positive Wet and slow moving.
CAM 1 Positive
CAM 2 Positive
TET 1 Positive
TET 2 Positive
Control (water) Positive

The results we got for antibiotics testing show that biofilms can still be recultured even after being treated with antibiotics.

Testing with cleaners

Cleaner Results After
Bleach -
Tilex 3
Mr. Clean -
Fantastik 1
Truly 2
Windex 1
Lysol -
GreenWorks 1
Biofilms fluorescent imaging under Typhoon
Different concentration of oxalic acid 3 minutes into incubation time at RT
E. coli cultures under varying concentration of oxalic acid
E. coli cultures under varying concentration of oxalic acid
Microcentrifuge tube containing Honey Bee
Microcentrifuge tube containing Varroa mites