|Skory, Christopher - Chris|
Submitted to: Fungal Genetics Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 3/23/2003
Publication Date: 3/23/2003
Citation: RAI, G., RAYAPATI, J., TONUKARI, J., SKORY, C.D. TRANSFORMATION OF ENZYME-TREATED RHIZOPUS ORYZAE GERMLINGS BY SQUARE-WAVE ELECTROPORATION. FUNGAL GENETICS CONFERENCE. 2003. ABSTRACT NO. 467.
Technical Abstract: Transformation involves the introduction of foreign DNA molecule into a cell for investigating gene structure and function. While bacterial transformations are relatively simple, eukaryotic transformations are always associated with problems that differ greatly between the transforming organisms. This necessitates optimization of complex variables, thereby resulting in a wide array of published transformation protocols available for eukaryotes. These transformation protocols can vary significantly from one species to another, although they can be usually adaptable to organisms within a species. In fungi, the spheroplasts generated from mycelia or spores are routinely transformed by chemical methods or by electroporation. Protoplasts have also been successfully transformed, but this procedure requires tedious optimization and is quite laborious. In some fungal species, intact spores have also been transformed with foreign DNA molecules, albeit with limited success and efficiencies. The biolistic transformation method has proved very helpful in species, including fungi, that have been recalcitrant to the conventional means. The use of this microprojectile bombardment method has been limited because of its cost. We report here a transformation procedure for the partially digested germlings of the filamentous fungus, Rhizopus oryzae. This protocol is relatively simple and time efficient. An overnight culture of spores was washed and further incubated for 2-4 hours with a mixture of lysing enzymes containing chitinase, chitosanase, and zymolyase. The mixture of partially digested germlings and DNA was electroporated in a BTX square-wave electroporator. The transforming DNA consisted of a circular plasmid with a functional lactate dehydrogenase and the pyrG genes under control of their respective promoter and terminator sequences. The transformed cells lacked the functional pyrG gene thus allowing selection by functional complementation on minimal plates lacking uracil.