Difference between revisions of "Team:Tec-Chihuahua"

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<p align="justify"> American and European Foulbrood are diseases affecting honey bee (Apis mellifera) larvae all around the world. The causal agents of these are two gram-positive bacteria: Paenibacillus larvae and Melissococcus plutonius. Nowadays, two techniques for the treatment of AFB and EFB are used: antibiotics and incineration of affected hives. The former promotes the development of antibiotic resistance in bacteria while the latter results unprofitable for beekeepers. The production of native bee antimicrobial peptides (AMPs) in Escherichia coli is proposed to treat P. larvae and M. plutonius infections. Defensin 1, abaecin, defensin 2, and apidaecin will each be expressed in a different culture. A 2⁴ factorial design will be used to identify the optimal AMP combination. The final product, AMPs with a specific packaging that can be released in the presence of Foulbrood, will be available for beekeepers to apply in their beehives and inhibit the proliferation of pathogenic bacteria. </p>  
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<p align="justify"> We suggest 4 AMPs for the treatment of <i> Melissococcus plutonius </i> and <i> Paenibacillus larvae </i>. Defensin 1, Defensin 2, Apidaecin and Abaecin are suggested AMPs. These AMPs possess different mechanisms of action. The defensins depolarize the membrane, open channels in it, and allow the efflux of potassium ions, which would either be compensated with contaminating cations or efflux of anions, destabilizing biochemical processes. There also is evidence that defensins stop respiratory activity and reduce the level of ATPs present. Abaecin and apidaecin, on the other hand, stop protein synthesis through interference of the 70S ribosome and inhibits DnaK activity. Apidaecin binds to LPS and disrupts the ABC transport system, as well.
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The project’s objective is to express these AMPs in a different bacterial culture each, with an inducible promoter. T7 RNA polymerase expression is regulated by the Lac operon, which is induced through the presence of IPTG. Therefore, the usage of T7 promoters will allow us to induce AMP production. Proteins will be extracted through sonication of bacterial cultures and then isolated through a His-tag purification process.
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Once all four AMPs are purified, all possible combinations will be tested against P. larvae and <i> M. plutonius </i>. A 2⁴ factorial design will be used to evaluate individual effects, quantify interactions, and identify the optimal AMP combination. PLGA-microencapsulated AMPs would be released in the bees’ diet so nurses can bring the AMPs to the infected larvae. This final product would be available for beekeepers to apply in their beehives through internal or external feeding mechanisms and thus inhibit the proliferation of the pathogenic bacteria.</p>  
 
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Revision as of 22:56, 20 June 2018

Erwinions

Bee a hero!

We suggest 4 AMPs for the treatment of Melissococcus plutonius and Paenibacillus larvae . Defensin 1, Defensin 2, Apidaecin and Abaecin are suggested AMPs. These AMPs possess different mechanisms of action. The defensins depolarize the membrane, open channels in it, and allow the efflux of potassium ions, which would either be compensated with contaminating cations or efflux of anions, destabilizing biochemical processes. There also is evidence that defensins stop respiratory activity and reduce the level of ATPs present. Abaecin and apidaecin, on the other hand, stop protein synthesis through interference of the 70S ribosome and inhibits DnaK activity. Apidaecin binds to LPS and disrupts the ABC transport system, as well. The project’s objective is to express these AMPs in a different bacterial culture each, with an inducible promoter. T7 RNA polymerase expression is regulated by the Lac operon, which is induced through the presence of IPTG. Therefore, the usage of T7 promoters will allow us to induce AMP production. Proteins will be extracted through sonication of bacterial cultures and then isolated through a His-tag purification process. Once all four AMPs are purified, all possible combinations will be tested against P. larvae and M. plutonius . A 2⁴ factorial design will be used to evaluate individual effects, quantify interactions, and identify the optimal AMP combination. PLGA-microencapsulated AMPs would be released in the bees’ diet so nurses can bring the AMPs to the infected larvae. This final product would be available for beekeepers to apply in their beehives through internal or external feeding mechanisms and thus inhibit the proliferation of the pathogenic bacteria.