Difference between revisions of "Team:Tec-Chihuahua/Results"
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<div class="a"><p align="justify">The following plan aims to test the efficiency of the produced antimicrobial peptides in the larvae considering characteristics of a real hive.</p></div> | <div class="a"><p align="justify">The following plan aims to test the efficiency of the produced antimicrobial peptides in the larvae considering characteristics of a real hive.</p></div> | ||
<ol> | <ol> | ||
| − | <li><u>Objective: Verify peptide expression</u>.<br>After inducing expression in BL21 (DE3) transformed with the gene of interest, the presence of peptides is verified through an SDS-Page analysis.</li> | + | <li><u>Objective: Verify peptide expression</u>.<br><div class="a">After inducing expression in BL21 (DE3) transformed with the gene of interest, the presence of peptides is verified through an SDS-Page analysis.</div></li><br> |
| − | <li><u>Objective: Obtain the purified peptides of interest</u>.<br>Technique of immobilized metal affinity chromatography (IMAC) is performed.</li> | + | |
| − | <li><u>Objective: Obtain the mature peptides of interest (without fusion proteins) purified</u>.<br>Digestion with enterokinase is performed and the mature peptide is purified employing ion exchange chromatography.</li> | + | <li><u>Objective: Obtain the purified peptides of interest</u>.<br><div class="a">Technique of immobilized metal affinity chromatography (IMAC) is performed.</div></li><br> |
| − | <li><u>Objective: Verify and quantify the inhibitory capacity of antimicrobial peptides against <i>Paenibacillus larvae </i>and <i>Melissococcus plutonius</i></u>.<br>Antibiograms are performed in a liquid medium with every possible peptide combination, and each peptide at two concentrations (24 factorial design experimentation) against <i>Paenibacillus larvae</i> and <i>Melissococcus plutonius</i> separately. Data is analyzed to prove the significance and effect of each factor.</li> | + | |
| − | <li><u>Objective: Confer the peptides protection that promotes their stability and preservation</u>.<br>After defining which peptide or combination of peptides is most efficient for the inhibition of bacteria, nanoencapsulation with PLGA of the chosen product is carried out.</li> | + | <li><u>Objective: Obtain the mature peptides of interest (without fusion proteins) purified</u>.<br><div class="a">Digestion with enterokinase is performed and the mature peptide is purified employing ion exchange chromatography.</div></li><br> |
| − | <li><u>Objective: Verify that the natural development of larvae is not altered after adding the treatment to the food</u>.<br>After the queen bee lays the eggs in each cell of the in vitro breeding place, proceeds to prepare and supply, with the help of a pipette, the feed of the larvae, containing the minimum inhibitory amount of the nano-encapsulated antimicrobial peptides obtained from the previous antibiogram tested against each bacterium. The same above procedure is followed with another cage and a homologous breeding place; in this case, the food will not contain the nanocapsules.</li> | + | |
| − | <li><u>Objective: Infect one larval community with <i>M. plutonius</i> and another with <i>P. larvae</i></u>.<br>Enough CFU are added to the feed of each cage, one with <i>M. plutonius</i> and the other with <i>P. larvae</i>.</li> | + | <li><u>Objective: Verify and quantify the inhibitory capacity of antimicrobial peptides against <i>Paenibacillus larvae </i>and <i>Melissococcus plutonius</i></u>.<br><div class="a">Antibiograms are performed in a liquid medium with every possible peptide combination, and each peptide at two concentrations (24 factorial design experimentation) against <i>Paenibacillus larvae</i> and <i>Melissococcus plutonius</i> separately. Data is analyzed to prove the significance and effect of each factor.</div></li><br> |
| − | <li><u>Objective: Verify and observe whether the treatment will diminish or cure the disease of the larvae</u>.<br>The food is mixed with the nanocapsules that are later supplied to the infected larvae. A cage with infected larvae that is provided with food without nanocapsules is considered.</li> | + | |
| − | <li><u>Objective: Verify that the nanocapsules reach the digestive tract of the larvae taking into account the real mechanism of a hive, where the nurse bees feed the larvae</u>.<br>A small nucleus is contemplated for the following experiment: Both nurse bees and larvae are found in the nucleus. The nanocapsules are placed in artificial liquid food and painted with FCF blue, (as suggested by Dr. Tanus). After 24-36 hours, a dissection of both the nurses and the larvae is performed in search for the nanocapsules. The extraction of hypopharyngeal glands in nurse bees serves as a checkpoint (as suggested by Dr. de Jong).</li> | + | <li><u>Objective: Confer the peptides protection that promotes their stability and preservation</u>.<br><div class="a">After defining which peptide or combination of peptides is most efficient for the inhibition of bacteria, nanoencapsulation with PLGA of the chosen product is carried out.</div></li><br> |
| − | <li><u>Objective: Verify that the treatment will diminish or cure the disease of the larvae in an environment cognate to the real environment of a hive; where the nurse bees feed the larvae</u>.<br>The previous experiment is repeated, but now the larvae are first infected; one nucleus with <i>P. larvae</i> and another with <i>M. plutonius</i>. Subsequently, the nanocapsules are added to the artificial food of the nucleus. The control consists of a nucleus where larvae are infected, but their artificial food is not supplied with nanocapsules.</li> | + | |
| − | <li><u>Objective: Grant the product the desired and appropriate characteristics for its sale and final application</u>.<br>Tests are made for the final presentation of the nanocapsules for the sale product.</li> | + | <li><u>Objective: Verify that the natural development of larvae is not altered after adding the treatment to the food</u>.<br><div class="a">After the queen bee lays the eggs in each cell of the in vitro breeding place, proceeds to prepare and supply, with the help of a pipette, the feed of the larvae, containing the minimum inhibitory amount of the nano-encapsulated antimicrobial peptides obtained from the previous antibiogram tested against each bacterium. The same above procedure is followed with another cage and a homologous breeding place; in this case, the food will not contain the nanocapsules.</div></li><br> |
| + | |||
| + | <li><u>Objective: Infect one larval community with <i>M. plutonius</i> and another with <i>P. larvae</i></u>.<br><div class="a">Enough CFU are added to the feed of each cage, one with <i>M. plutonius</i> and the other with <i>P. larvae</i>.</div></li><br> | ||
| + | |||
| + | <li><u>Objective: Verify and observe whether the treatment will diminish or cure the disease of the larvae</u>.<br><div class="a">The food is mixed with the nanocapsules that are later supplied to the infected larvae. A cage with infected larvae that is provided with food without nanocapsules is considered.</div></li><br> | ||
| + | |||
| + | <li><u>Objective: Verify that the nanocapsules reach the digestive tract of the larvae taking into account the real mechanism of a hive, where the nurse bees feed the larvae</u>.<br><div class="a">A small nucleus is contemplated for the following experiment: Both nurse bees and larvae are found in the nucleus. The nanocapsules are placed in artificial liquid food and painted with FCF blue, (as suggested by Dr. Tanus). After 24-36 hours, a dissection of both the nurses and the larvae is performed in the search for the nanocapsules. The extraction of hypopharyngeal glands in nurse bees serves as a checkpoint (as suggested by Dr. de Jong).</div></li><br> | ||
| + | |||
| + | <li><u>Objective: Verify that the treatment will diminish or cure the disease of the larvae in an environment cognate to the real environment of a hive; where the nurse bees feed the larvae</u>.<br><div class="a">The previous experiment is repeated, but now the larvae are first infected; one nucleus with <i>P. larvae</i> and another with <i>M. plutonius</i>. Subsequently, the nanocapsules are added to the artificial food of the nucleus. The control consists of a nucleus where larvae are infected, but their artificial food is not supplied with nanocapsules.</div></li><br> | ||
| + | |||
| + | <li><u>Objective: Grant the product the desired and appropriate characteristics for its sale and final application</u>.<br><div class="a">Tests are made for the final presentation of the nanocapsules for the sale product.</div></li> | ||
</ol><br> | </ol><br> | ||
</div> | </div> | ||
Revision as of 07:08, 15 October 2018
Successes & Failures
The iGEM experience taught us a lot of unique things, and as we had significant achievements, we went through many unexpected obstacles. Throughout the year we faced many challenges for finally reaching our goal; we believe that the effort was worthwhile.
Model
Successful
With minimal mathematical and informatics background, we were able to adapt a model to our situation and code effective solutions for data analysis. This model can also help further developments in similar projects.
Unsuccessful
Our mathematical model went through many iterations (no pun intended) before arriving at its final state: we tried to develop a molecular model to evaluate protein production and a populational model to predict the health of the beehive by eliminating nodes of infection. In the end, time and resources forced us to concentrate our efforts on the most pressing of matters: peptide delivery.
We were unsuccessful in solving the equation for D; we tried several series properties and other sorts of tricks, but we couldn’t find an analytical solution. We decided to find the diffusion coefficient with a simulation of multiple lines.
Our most poignant failure was not being able to run the experiments successfully. Apidaecin was retained at a customs office for a whole month. By the time we needed the nanoencapsulation to be at the lab for experimentation, the peptide had not yet arrived. We are left with a burning vigor to help make importation easier for all sorts of experimentation in Mexico.
Laboratory
Successful
We are the first iGEM team from Tec-Chihuahua that manages to send successful results for the InterLab Study. Besides being a rewarding experience, it was a lot of learning since we had never used a microplate reader.
Unsuccessful
At the beginning, we worked with E. coli TOP10 but later we had to change to DH5a since the TOP10 strain was contaminated.
For a few weeks, the successful transformation of our competent cells was difficult. We discovered that the key was in the quality of the competent cells. We tried to make several stocks of competent cells with glycerol and store them at -20 °C, however, this did not work. We had to make the competent cells on the same day that the transformation protocol was to be carried out.
We worked in the transformation of competent cells of DH5a with the synthesis of our part linked with pSB1C3. When they were disseminated in the LB medium with chloramphenicol, there was a presence of colonies which meant that they had been correctly transformed. However, when performing the electrophoresis of plasmid extraction only one band of the size of pSB1C3 was observed. First, we thought that the protocols of digestion and ligation that we carried out were not the correct ones or there was a bad manipulation. This idea was discarded when using a control with RFP. Doing more research, we discovered that the parts that we sent to synthesize in IDT lacked base pairs next to the restriction sites of EcoRI-HF and PstI. This means that the enzyme has no way to attach to the DNA sequence and digest it. Why did we have colonies that grew on LB + CAM? We believe that the re-circularization of the plasmid was possible.
Future Plans
Eight months are not enough to address the full scope of our project. This does not end here!
Experimentation Plan
The following plan aims to test the efficiency of the produced antimicrobial peptides in the larvae considering characteristics of a real hive.
- Objective: Verify peptide expression.After inducing expression in BL21 (DE3) transformed with the gene of interest, the presence of peptides is verified through an SDS-Page analysis.
- Objective: Obtain the purified peptides of interest.Technique of immobilized metal affinity chromatography (IMAC) is performed.
- Objective: Obtain the mature peptides of interest (without fusion proteins) purified.Digestion with enterokinase is performed and the mature peptide is purified employing ion exchange chromatography.
- Objective: Verify and quantify the inhibitory capacity of antimicrobial peptides against Paenibacillus larvae and Melissococcus plutonius.Antibiograms are performed in a liquid medium with every possible peptide combination, and each peptide at two concentrations (24 factorial design experimentation) against Paenibacillus larvae and Melissococcus plutonius separately. Data is analyzed to prove the significance and effect of each factor.
- Objective: Confer the peptides protection that promotes their stability and preservation.After defining which peptide or combination of peptides is most efficient for the inhibition of bacteria, nanoencapsulation with PLGA of the chosen product is carried out.
- Objective: Verify that the natural development of larvae is not altered after adding the treatment to the food.After the queen bee lays the eggs in each cell of the in vitro breeding place, proceeds to prepare and supply, with the help of a pipette, the feed of the larvae, containing the minimum inhibitory amount of the nano-encapsulated antimicrobial peptides obtained from the previous antibiogram tested against each bacterium. The same above procedure is followed with another cage and a homologous breeding place; in this case, the food will not contain the nanocapsules.
- Objective: Infect one larval community with M. plutonius and another with P. larvae.Enough CFU are added to the feed of each cage, one with M. plutonius and the other with P. larvae.
- Objective: Verify and observe whether the treatment will diminish or cure the disease of the larvae.The food is mixed with the nanocapsules that are later supplied to the infected larvae. A cage with infected larvae that is provided with food without nanocapsules is considered.
- Objective: Verify that the nanocapsules reach the digestive tract of the larvae taking into account the real mechanism of a hive, where the nurse bees feed the larvae.A small nucleus is contemplated for the following experiment: Both nurse bees and larvae are found in the nucleus. The nanocapsules are placed in artificial liquid food and painted with FCF blue, (as suggested by Dr. Tanus). After 24-36 hours, a dissection of both the nurses and the larvae is performed in the search for the nanocapsules. The extraction of hypopharyngeal glands in nurse bees serves as a checkpoint (as suggested by Dr. de Jong).
- Objective: Verify that the treatment will diminish or cure the disease of the larvae in an environment cognate to the real environment of a hive; where the nurse bees feed the larvae.The previous experiment is repeated, but now the larvae are first infected; one nucleus with P. larvae and another with M. plutonius. Subsequently, the nanocapsules are added to the artificial food of the nucleus. The control consists of a nucleus where larvae are infected, but their artificial food is not supplied with nanocapsules.
- Objective: Grant the product the desired and appropriate characteristics for its sale and final application.Tests are made for the final presentation of the nanocapsules for the sale product.
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