Agar plate freezing assay for the in situ selection of transformed ice nucleating bacteria.
An agar plate freezing assay is described based on the incorporation of fluorescein dye in agar medium. Upon addition of fluorescein the medium becomes transparent. This facilitates the monitoring of the ice nucleation event in vivo and the subsequent in situ selection of transformed ice nucleating bacteria. In comparison with known assays for the screening of transformants, the proposed assay is very accurate and reproducible. It may be applied in environmental samples screening for ice nucleating organisms, or in cDNA or genomic libraries for identifying novel ice nucleation genes. It may also prove useful in comparative studies of the ice nucleation activity, e.g. in directed evolution experiments involving ice nucleation genes.
Anastassopoulos
Elias
E
TEI Larissas, School of Agricultural Technology, Department of Plant Production, Perifereiaki Odos Trikalon Karditsas, 41335 Larissa, Greece. anastaso@teilar.gr
D000362:Agar / Q000494:pharmacology*; D021301:Antifreeze Proteins / Q000737:chemistry; D001419:Bacteria / Q000737:chemistry / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D019793:Fluorescein / Q000494:pharmacology; D005615:Freezing; D015698:Genomic Library; D007053:Ice; D008856:Microscopy, Fluorescence / Q000379:methods
D021301:Antifreeze Proteins; D001425:Bacterial Outer Membrane Proteins; D003451:Cryoprotective Agents; D018076:DNA, Complementary; D007053:Ice; C063113:ice nucleation protein; D000362:Agar; D019793:Fluorescein