FREEZING ASSAY FOR THE SELECTION OF ICE NUCLEATING ORGANISMS AND THE CLONING OF ICE NUCLEATIONS GENES

摘要

The invention pertains to a method for the selection of ice nucleating organisms and the cloning of ice nucleation genes. The method comprising (a) preparing a transparent solid, or semisolid growth medium, (b) inoculating the transparent medium with ice nucleating organisms, (c) incubating plates at appropriate temperature and for the appropriate time period for the growth of the inoculated organism, (d) placing the plates to float on the surface of an alcohol bath, maintained at or below zerotemperatures, (e) monitoring of the ice nucleation event in vivo, visually or by recording e.g. by a video camera, and (f) subsequent in situ selection of ice nucleating organisms, based on the change of the opacity of the medium, caused during and after freezing. The method of the present invention further comprises the method described above wherein the transparent growth medium is prepared by adding fluorescein or any other substance that renders the medium transparent. The method of the present invention further comprises the method described above wherein the method is applied in environmental samples screening for ice nucleating organisms. The method of the present invention further comprises the method described above wherein the method is used for cloning novel ice nucleation genes, or for cloning novel genes, with the use of ice nucleation reporter molecules. The method of the present invention further comprises the method described above wherein the method is applied in cDNAor genomic libraries. The method of the present invention further comprises the method described above wherein the method is used for ice nucleation studies, including a) comparative studies of the ice nucleation activity, either between ice nucleation genes, or between ice nucleating organisms, b) mutational studies or c) directed evolution studies, involving ice nucleation genes, d) identifying factors involved in the assembly of the ice nucleation proteins...