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

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<h1 id="projecttext1" class="contentSubTitle">Testing our construct<br><small></small></h1>
 
<h1 id="projecttext1" class="contentSubTitle">Testing our construct<br><small></small></h1>
  
 +
<h1 id="projecttext1" class="contentSubTitle">Biofilms experiments and protocols<br><small></small></h1>
 +
<p id="humanpractices_hp" class="bees"><b>Growing biofilms</b><br></p>
 +
<p>In the lab for biofilms, our first focus was on trying to see if our team could grow biofilms. We successfully did this using the following protocol:</p>
 +
<ol>
 +
<li>In a 50 mL falcon tube, 21 mL of LB media, 210 µL of RFP, and 21 μL of KAN were added. This solution was then mixed more thoroughly by putting the cap on the falcon tube and inverting it a few times.</li>
 +
<li>3 mL of this solution was pipetted into seven 35x10 mm Petri dishes. </li>
 +
<li>The Petri dishes were then labeled and put into an incubator for 24 hours at 37℃.</li>
 +
<li>After 24 hours, the solution was poured out of each dish into a bleach container. The dishes were then rinsed with distilled water twice.</li>
 +
<li>___ drops of crystal violet were placed around the rim of the Petri dishes. If a biofilm had formed, a ring of crystal violet would be seen. </li>
  
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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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</ol>
  
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<b>What is nuclease? What is dextranase?</b><br>
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<p id="humanpractices_hp" class="bees"><b>Testing with home cleaners:</b><br></p>
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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>
+
<p>Once we knew that we could grow biofilms, we decided to test out the effectiveness of home cleaning products. The products we tested are Mr. Clean, Fantastik, Truly, Windex, Tilex, Bleach, Lysol, and GreenWorks. The protocol that we used for this is as follows:</p>
−
+
<ol>
−
<b>What we are doing differently</b><br>
+
<li>Using biofilms made the previous day with protocol 1, the experimenter removed the contents in the dish in the bleach container. Then the experimenter rinsed the Petri dish with distilled water twice. The Petri dishes were laid out on paper towel to let the excess water drip out.</li>
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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>
+
<li>After the Petri dishes were thoroughly dry, 3.2 mL of one of the listed cleaning products was pipetted into a Petri dish. The cleaning product was left in the Petri dish for 30 minutes (If bleach was being used, only for 15 minutes).</li>
 +
<li>The cleaning product was then disposed of in the bleach container. The Petri dish was then rinsed with water once. </li>
 +
<li>To see if a biofilm was still remaining, 100 μL of LB media was pipetted onto the inside edge of the Petri dish.</li>
 +
<li>The LB media was then pipetted back into the LB media tube along with 5 μL of KAN.</li>
 +
<li>This was then put into the shaker for 24 hours at 37 ℃.</li>
  
−
<b>Extracellular Polymeric Substance Matrix: Sticky Stuff!</b><br>
+
</ol>
−
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.
+
<p id="humanpractices_hp" class="biofilms"><b>Testing Antibiotics:</b><br></p>
−
</p>
+
<p>We also tested antibiotics to see if they could get rid of biofilms as well. We used this protocol:
 +
</p>
 +
<ol>
 +
<li>Biofilms made using protocol 1 were rinsed twice using distilled water. </li>
 +
<li>In twelve 2 mL tubes, 1.65 mL of double distilled water was pipetted into each of them. Into two 2 mL tubes, 3.3 μL of AMP was pipetted into them.
 +
<li>Step 2 was repeated using CAM, TET, and water as a control.</li>
 +
<li>Two of the 2 mL tubes for each of the antibiotic and control 2 mL tubes were each placed into a 35x10 mm Petri dish. This was repeated with five other Petri dishes. </li>
 +
<li>Three of them were rinsed after 15 minutes while the other three and the control were rinsed after 30 minutes. </li>
 +
<li>These were then recultured by pipetting 100 μL of LB media into the rim of the 35x10 mm Petri dish. This was then pipetted back into the tube along with 5 μL of KAN.</li>
 +
<li>The Petri dishes were then put into an incubator for 24 hours at 37 ℃.</li>
  
 +
</ol>
 +
 +
<p id="humanpractices_hp" class="biofilms"><b>Typhoon:</b><br></p>
 +
<p>To get quantitative data, we used the Typhoon.
 +
</p>
 +
<ol>
 +
<li>An area on the grid of the typhoon was chosen depending on how many 35x10 mm Petri dishes we used.</li>
 +
<li>Fluorescence scanning was chosen; the setting for that was 580 BP, AlexaFlour546, PMT: 600, and with a green laser.</li>
 +
<li>The pixel size used was 100 microns.</li>
 +
<li>The focal plane used was +3 mm so that it would scan through the plastic bottom of the Petri dish.</li>
 +
 +
</ol>
 
</section>
 
</section>
 
<section id="section2">
 
<section id="section2">

Revision as of 10:15, 18 September 2015

Project

Experiments

Bees experiments and protocols

Oxalic Acid Gradient:

  1. In 15 mL falcon tubes, 5 mL of sulfuric acid, 2 mL of Potassium Permanganate, and x amount of oxalic acid.
  2. The solutions would then sit for 10 minutes, being inverted every few minutes.
  3. The optical density with a wavelength of 528 nm was taken for each falcon tube. The results were recorded.

Mite Trials:

  1. Pouches were made using parafilm. This was done by stretching out the parafilm very thinly with the experimenter’s hands.
  2. Using a pen cap, the parafilm was arranged so that there was a small pouch at the bottom for the mite and solution to fit in.
  3. The pen was removed and the top of the parafilm was left open.
  4. A Varroa mite was then placed into the pouch along with a solution such as water.
  5. They were left overnight in the 27 ℃ incubator.

Bee Trials:

  1. Bees were obtained from a bee hive.
  2. The bees were then fed RFP producing E.coli with glucose and sucrose sugars. The bees were then observed to determine if they were drinking.
  3. Mites were added to Petri dishes following a sugar shake. Paper derivatives were soaked in different treatments , water and RFP E.coli and were in the bottom of the Petri dishes.
  4. The Petri dishes were then placed into the incubator for 24 hours at 27 ℃.
  5. The bees were euthanized using EtOH, surface sterilized in bleach, opened using a razor to expose the gut, and placed into the shaker overnight at 37℃.

      6. Testing our construct

      Biofilms experiments and protocols

      Growing biofilms

      In the lab for biofilms, our first focus was on trying to see if our team could grow biofilms. We successfully did this using the following protocol:

      1. In a 50 mL falcon tube, 21 mL of LB media, 210 µL of RFP, and 21 μL of KAN were added. This solution was then mixed more thoroughly by putting the cap on the falcon tube and inverting it a few times.
      2. 3 mL of this solution was pipetted into seven 35x10 mm Petri dishes.
      3. The Petri dishes were then labeled and put into an incubator for 24 hours at 37℃.
      4. After 24 hours, the solution was poured out of each dish into a bleach container. The dishes were then rinsed with distilled water twice.
      5. ___ drops of crystal violet were placed around the rim of the Petri dishes. If a biofilm had formed, a ring of crystal violet would be seen.

      Testing with home cleaners:

      Once we knew that we could grow biofilms, we decided to test out the effectiveness of home cleaning products. The products we tested are Mr. Clean, Fantastik, Truly, Windex, Tilex, Bleach, Lysol, and GreenWorks. The protocol that we used for this is as follows:

      1. Using biofilms made the previous day with protocol 1, the experimenter removed the contents in the dish in the bleach container. Then the experimenter rinsed the Petri dish with distilled water twice. The Petri dishes were laid out on paper towel to let the excess water drip out.
      2. After the Petri dishes were thoroughly dry, 3.2 mL of one of the listed cleaning products was pipetted into a Petri dish. The cleaning product was left in the Petri dish for 30 minutes (If bleach was being used, only for 15 minutes).
      3. The cleaning product was then disposed of in the bleach container. The Petri dish was then rinsed with water once.
      4. To see if a biofilm was still remaining, 100 μL of LB media was pipetted onto the inside edge of the Petri dish.
      5. The LB media was then pipetted back into the LB media tube along with 5 μL of KAN.
      6. This was then put into the shaker for 24 hours at 37 ℃.

      Testing Antibiotics:

      We also tested antibiotics to see if they could get rid of biofilms as well. We used this protocol:

      1. Biofilms made using protocol 1 were rinsed twice using distilled water.
      2. In twelve 2 mL tubes, 1.65 mL of double distilled water was pipetted into each of them. Into two 2 mL tubes, 3.3 μL of AMP was pipetted into them.
      3. Step 2 was repeated using CAM, TET, and water as a control.
      4. Two of the 2 mL tubes for each of the antibiotic and control 2 mL tubes were each placed into a 35x10 mm Petri dish. This was repeated with five other Petri dishes.
      5. Three of them were rinsed after 15 minutes while the other three and the control were rinsed after 30 minutes.
      6. These were then recultured by pipetting 100 μL of LB media into the rim of the 35x10 mm Petri dish. This was then pipetted back into the tube along with 5 μL of KAN.
      7. The Petri dishes were then put into an incubator for 24 hours at 37 ℃.

      Typhoon:

      To get quantitative data, we used the Typhoon.

      1. An area on the grid of the typhoon was chosen depending on how many 35x10 mm Petri dishes we used.
      2. Fluorescence scanning was chosen; the setting for that was 580 BP, AlexaFlour546, PMT: 600, and with a green laser.
      3. The pixel size used was 100 microns.
      4. The focal plane used was +3 mm so that it would scan through the plastic bottom of the Petri dish.