Team:UANL Mty-Mexico/Safety/P

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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.

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