Ice-minus Bacteria - Production

Production

To systematically create the ice-minus strain of P. syringae, its ice-forming gene must be isolated, amplified, deactivated and reintroduced into P. syringae bacterium. The following steps are often used to isolate and generate ice-minus strains of P. syringae:

1. Digest P. syringae's DNA with restriction enzymes.
2. Insert the individual DNA pieces into a plasmid. Pieces will insert randomly, allowing for different variations of recombinant DNA to be produced.
3. Transform the bacterium Escherichia coli (E.coli) with the recombinant plasmid. The plasmid will be taken in by the bacteria, rendering it part of the organism's DNA.
4. Identify the ice-gene from the numerous newly developed E. coli recombinants. Recombinant E. coli with the ice-gene will possess the ice-nucleating phenotype, these will be "ice-plus."
5. With the ice nucleating recombinant identified, amplify the ice gene with techniques such as polymerase chain reactions (PCR).
6. Create mutant clones of the ice gene through the introduction of mutagenic agents such as UV radiation to inactivate the ice gene, creating the "ice-minus" gene.
7. Repeat previous steps (insert gene into plasmid, transform E. coli, identify recombinants) with the newly created mutant clones to identify the bacteria with the ice-minus gene. They will possess the desired ice-minus phenotype.
8. Insert the ice-minus gene into normal, ice-plus P. syringae bacterium.
9. Allow recombination to take place, rendering both ice-minus and ice-plus strains of P. syringae.