Team:Cornell/project/wetlab/fungal toolkit/selectable markers

Antibiotic resistance is an important component of most molecular cloning; allowing for selection of transformants by killing or inhibiting untransformed or incompletely transformed cells greatly facilitates finding successful transformants. We found three antibiotics that are effective against basidiomycetes and their resistance genes: hygromycin (hph), geneticin (nptII), and phosphinothricin (bar) . We attempted to develop as many configurations of these resistances as possible, putting them behind a variety of promoters, and removing any RFC 10 cut sites that exist in the resistance genes.


We used the T7 promoter, the promoters for trpC and gpdA in Aspergillus nidulans, and the gpdA promoter from Ganoderma lucidum. For trpC, there is also a terminator, which we appended to PtrpC constructs. The T7 promoter will allow these resistances to be used in orthogonal expression systems, and facilitate transformation of large constructs with several promoters by greatly reducing promoter length. The Aspergillus nidulans promoters should provide high constitutive expression for ascomycetes, and low expression for basidiomycetes, while the Ganoderma lucidum PgpdA provides high constitutive expression for basidiomycetes, and should provide low expression for ascomycetes [1].
We transformed the construct pC13BV (T7 promoter and hygromycin resistance gene) into the BL21-A1 strain of E. coli, and measured the effects of hygromycin on the wild type and plasmid-containing cells. The diameters of inhibition were statistically significantly different (using a one-tailed student's t-test, with an alpha value of 0.005), demonstrating the functionality of our T7 promoter and hygromycin resistance construct.

References

1. Amir Sharon (ed.), Molecular and Cell Biology Methods for Fungi, Methods in Molecular Biology, vol. 638. Turgeon BG, Condon B, Liu J, Zhang N (2010). Protoplast Transformation of Filamentous Fungi.