Ice Minus Experiment

THE BEGININGS • It is invented by STEVEN LINDOW. He is the plant pathologist, discovered that the bacterium PSEUDOMONAS SYRINGAE is one of the more prevalent ice forming organisms in nature. • This technique is successfully applied only in NORTH AMERICA.

ICE MINUS BACTERIA Gene altered bacteria unable to produce the proteins that participate in the crystal formation ICE NUCLEATING BACTERIA Bacteria that synthesize proteins, which blend with water to form ice crystals at 32°F.

DEFINITION:Ice-minus bacteria is a nickname given to a variant of the common bacterium Pseudomonas syringae (P. syringae). This strain of P. syringae lacks the ability to produce a certain surface protein, usually found on wild-type "ice-plus" P. syringae. The "ice-plus" protein (Ina protein, "Ice nucleation-active" protein) found on the outer bacterial cell wall acts as the nucleating centers for ice crystals. This facilitates ice formation, hence the designation "ice-plus." The ice-minus variant of P. syringae is a mutant, lacking the gene responsible for ice-nucleating surface protein production.

STORY OF ICE-MINUS BACTERIA:Pseudomonas syringae • a bacterium lives on leaves and stems • protein on bacterial cell surface encourages ice crystal to form even when the air temperature is several degrees above freezing • makes many plants susceptible to frost damage

PRODUCTION:• Digest P. syringae's DNA with restriction enzymes • Insert the individual DNA pieces into a plasmid. Pieces will insert randomly, allowing for different variations of recombinant DNA to be produced. • 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.

• Identify the ice-gene from the numerous newly developed E. coli recombinants. Recombinant E. coli with the icegene will possess the ice-nucleating phenotype, these will be "ice-plus.“ • With the ice nucleating recombinant identified, amplify the ice gene with techniques such as polymerase chain reactions (PCR). • 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.

• Repeat previous steps (insert gene into plasmid, transform E. coli, identify recombinants) with the newly created mutant clones to identify the bacteria with the iceminus gene. They will possess the desired ice-minus phenotype • Insert the ice-minus gene into normal, iceplus P. syringae bacterium. • Allow recombination to take place, rendering both ice-minus and ice-plus strains of P. syringae.

ECONOMIC IMPORTANCE:• In the United States alone, it has been estimated that frost accounts for approximately $1 billion in crop damage each year (Before introduction of Ice minus bacteria) .