Team:UANL Mty-Mexico/Safety/P

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<p align="justify"><b>BIOCIENCIA BIOSECURITY PROCESS</b><br><br>In order to learn about biosecurity processes in companies related with our project, we visited BioCiencia S.A de C.V, a private company where, molecular biologists and agronomists, parasite engineers work, they have extensive experience, theoretical and in practice with national agriculture problems. They have a traditional diagnostic laboratory and a molecular plant where they are able to detect pathogens (caused by fungi, bacteria, viruses, viroids, nematodes and Mycoplasmas), using the most advanced technologies at a national and international level such as ELISA, PCR (Polymerase Chain Reaction), RT-PCR (reverse transcription-PCR), Immuno-capture RT-PCR and hybridization of nucleic acids with no radioactive probes, among others. Their laboratory is approved by SAGARPA, (Secretaría de agricultura, ganadería, desarrollo rural, pesca y alimentación) (Key approval no. 98-719 -001-F).<br><br>It was a pleasant surprise to discover that, a company that uses molecular biology in the detection of plant diseases was in our community. Their function is closely related to our project because while we develop a bioinseticide to control the plague Premnotrypesvorax, that attacks potatoe corps; they are part of the 13 plant pathology and entomologyindustries in México that check and approve fruit quality, vegetables and legumes, that are put in supermarkets to be sold.<br><br>BioCiencia was very accessible company, when they let us visit their laboratories. They were very at the moment of showing their laboratories and processes; they also explained the security standards that their installations need to satisfy in order to be approved as an official SAGARPA laboratory. Talking about the diagnostic process, first they receive phytosanitary samples that previously fulfilled necessary requirements to enter the laboratory, without taking into account if they are sick or healthy. Then, the samples are passed to the third part of the laboratory, which is divided in three other parts, the sample stays there until they are processed, liquefied or macerated. The samples are passed to the ISO format to have a better organization. After that they make the diagnose to see what kind of plague, bacteria or virus they are dealing with, that is directly affecting the plants health, they do this by two methods, the most common, PCR, for bacteria and virus cultures, and ISA, for the detection of proteins. All the culture media is sterilized.<br><br>Biosecurity is defined as the set of preventive measures designed to maintain control of occupational risk factors from biological, physical or chemical issues, preventing harmful impacts. The importance of security in the laboratory goes farther, because a failure in the process can cause the loss of the material and the whole work. To preventing this and in case of the client wanting a review on the product, the laboratory keeps samples for 30 days.<br><br>this area of work has a great impact, because it reduces the risk of new plagues to enter national territory. In order to be certificated by the SAGARPA and EMA, Mexican Accreditation Entity, the installations are cheeked each year up to a 40% and each four years to a full 100%. Besides fulfilling the basic requirements of every laboratory, as the usage of appropriate safety equipment and following the registry on each instrument, it has to satisfy the requirements of the ISO/IEC 17025, the Mexican Institute of Standardization and Certification A.C., and the general requirements for the competence of testing and calibration laboratories, that is the main ISO/CASCO standard used by testing and calibration laboratories. The contents of ISO/IEC 17025 standard itself with five elements which are: Scope, Normative References, Terms and Definitions, Management Requirements and Technical Requirements. The two main sections in ISO/IEC 17025 are Management Requirements and Technical Requirements. Management requirements are primarily related to the operation and effectiveness of the quality management system within the laboratory. Technical requirements include factors which determine the correctness and reliability of the tests and calibrations performed in laboratory.<br><br>Laboratories use ISO/IEC 17025 to have the standardized quality system aimed at improving their ability to consistently produce valid results. In the case of BioCiencia, the material they use is obtained from AGDIA, the World Leader in Plant Pathogen Test Kits, and NEOGEN. And all the results obtained from the studies they make, are put monthly in a DVD; they also need to have a common server and a hard disk drive, which, depending on the importance of the document, is the amount of people that has access to it. Noteworthy that all the staff working in BioCiencia has at least a master’s degree.  Another important point of this company is, that the results from the plants diagnostic, are strictly confidential to the customer. <br><br>The visit to BioCiencia made us realize the importance of having an effective security process, as well as the fulfillment of its normativity, as an essential part of the general process. If both are not satisfied, neither will be completed acquired, thus we cannot consider any kind of process complete. Security optimizes the process, time, materials, human resources, and reduces the margin of error inside the laboratory. We realized that for our own project we will need to follow what we learned in this visit in order to continue improving our work.</p><p><b>BioCiencia website:</b> http://www.biociencia.com.mx/inicio.html<br><b>General Director:</b> Dr.Ramiro Gonzalez Garza</p>
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<p align="justify"><div class="Estilo8"><b>SAFETY PROPOSAL</b></div><br><br>
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<b> Determine the specificity of the phage to the multiple strains of the genre E. coli, Pseudomonas, Clavibacter, Serratia and Bacillus</b><br><br>
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Determination of the host range of the manipulated bacteriophage is therefore an important step in the risk assessment process to evaluate the probability of the phage’s propagation in a particular environment and its potential role in global gene transfer. Phage P1 are generally specific to one type of bacteria, but this host-specificity can be changed or expanded to other bacterial species. Restriction/modification systems may also be important parameters that affect and limit the host range of a phage in some bacterial strains. One way to address this particular problem is by engineering phages with genomes that do not contain restriction sites recognized by the bacterial host.<br><br>
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<u>PROTOCOL: To determine the specificity of bacteriophage perform the following: </u><br>
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<ul><li>An aliquot of liquid culture to evaluate strains (E. coli K-12: Top10, DH10B, DH5α, DB3.1, Pseudomonas spp, Clavibacter spp, Bacillus subtilis B479, Pseudomonas syringe, Serrate spp) the OD is measured between 0.2 and 0.4, after obtaining this O.D., two aliquots of 300 uL of each strain in a 96-well plate will be arranged. </li><br>
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<li>To evaluate the susceptibility one of the aliquots were taken as a control and to the other aliquot add 50 uL of the bacteriophage P1 to the different strains to be evaluated, incubate for 1.5 h at 150 rpm, at 37ºC. </li><br>
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<li>Measure the O.D. strains every 30 minutes until the incubation time is completed.</li><br><br>
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<b> Determine phage viability under various conditions of pH, temperature and medium</b><br><br>
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Some phages can survive outside their microbial hosts for long periods of time under certain circumstances and maintain their ability to infect bacterial hosts. The survival and persistence of manipulated phages in soil or in other environments should therefore be studied carefully to evaluate the extent of potential risks in case of release.<br>
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<u> PROTOCOL: Determining the viability of bacteriophage P1 in various states of stress. </u><br>
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<ul><li>a) Stress of pH to determine the viability of pH stress</li>
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Phage be in liquid LB medium adjusted to different pH ranges; acid 4-6, optimum 7 and basic 8-10, then incubate overnight at 37ºC.<br>
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After incubation of virus, count CFU of E.coli MC1061 and incubate at 37°C.<br>
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This way you will be measured if pH stress influences the viability of the virus in the process of infection. <br><br>
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<li>b) Temperature stress</l><br>
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The phage grown in LB medium and incubated at different temperature ranges: (32ºC to 42oC.) For overnight. <br>
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After incubation of virus, count CFU of E.coli MC1061 and incubate at 37°C. <br>
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This way you will be measured if pH stress influences the viability of the virus in the process of infection.<br><br>
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<li>c) Nutrient stress</li>
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The phage grown in LB medium to be incubated at 37ºC. <br>
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After incubation of virus, count CFU of E.coli MC1061, which are hatched in different media (LB agar, M9 agar, LB agar supplemented with glycerol), and incubate at 37ºC. <br>
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This will measure whether nutrient stress affects the viability of the virus in the process of infection.</ul><br><br>
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<b> Determine potential multiple targets for the recognition sites of TALEN</b><br><br>
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With the BLAST tool from NCBI we are able to compare the recognition sites of TALEN in multiple genomes, basically divided in groups: Bacteria, Eukarya, Mammals and Human.<br>
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Latest revision as of 03:32, 18 October 2014

Safety
Proposal

SAFETY PROPOSAL


Determine the specificity of the phage to the multiple strains of the genre E. coli, Pseudomonas, Clavibacter, Serratia and Bacillus

Determination of the host range of the manipulated bacteriophage is therefore an important step in the risk assessment process to evaluate the probability of the phage’s propagation in a particular environment and its potential role in global gene transfer. Phage P1 are generally specific to one type of bacteria, but this host-specificity can be changed or expanded to other bacterial species. Restriction/modification systems may also be important parameters that affect and limit the host range of a phage in some bacterial strains. One way to address this particular problem is by engineering phages with genomes that do not contain restriction sites recognized by the bacterial host.

PROTOCOL: To determine the specificity of bacteriophage perform the following:
  • An aliquot of liquid culture to evaluate strains (E. coli K-12: Top10, DH10B, DH5α, DB3.1, Pseudomonas spp, Clavibacter spp, Bacillus subtilis B479, Pseudomonas syringe, Serrate spp) the OD is measured between 0.2 and 0.4, after obtaining this O.D., two aliquots of 300 uL of each strain in a 96-well plate will be arranged.

  • To evaluate the susceptibility one of the aliquots were taken as a control and to the other aliquot add 50 uL of the bacteriophage P1 to the different strains to be evaluated, incubate for 1.5 h at 150 rpm, at 37ºC.

  • Measure the O.D. strains every 30 minutes until the incubation time is completed.


  • Determine phage viability under various conditions of pH, temperature and medium

    Some phages can survive outside their microbial hosts for long periods of time under certain circumstances and maintain their ability to infect bacterial hosts. The survival and persistence of manipulated phages in soil or in other environments should therefore be studied carefully to evaluate the extent of potential risks in case of release.
    PROTOCOL: Determining the viability of bacteriophage P1 in various states of stress.
    • a) Stress of pH to determine the viability of pH stress
    • Phage be in liquid LB medium adjusted to different pH ranges; acid 4-6, optimum 7 and basic 8-10, then incubate overnight at 37ºC.
      After incubation of virus, count CFU of E.coli MC1061 and incubate at 37°C.
      This way you will be measured if pH stress influences the viability of the virus in the process of infection.

    • b) Temperature stress
      The phage grown in LB medium and incubated at different temperature ranges: (32ºC to 42oC.) For overnight.
      After incubation of virus, count CFU of E.coli MC1061 and incubate at 37°C.
      This way you will be measured if pH stress influences the viability of the virus in the process of infection.

    • c) Nutrient stress
    • The phage grown in LB medium to be incubated at 37ºC.
      After incubation of virus, count CFU of E.coli MC1061, which are hatched in different media (LB agar, M9 agar, LB agar supplemented with glycerol), and incubate at 37ºC.
      This will measure whether nutrient stress affects the viability of the virus in the process of infection.


    Determine potential multiple targets for the recognition sites of TALEN

    With the BLAST tool from NCBI we are able to compare the recognition sites of TALEN in multiple genomes, basically divided in groups: Bacteria, Eukarya, Mammals and Human.