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SAFETY PROPOSAL
Safety suggestions
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.
Determine the specificity of the phage to the multiple strains of the genre E. coli, Pseudomonas, Clavibacter, Serratia and Bacillus.
To determine the specificity of bacteriophage perform the following protocol:
- 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 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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