Cloning Fluorogen Activating Protein J10 VL for Protein Localization Studies in Candida Albicans

Fluorogen activating proteins (FAPs) are a new class of protein reporters that can generate fluorescence from otherwise non-fluorescent molecules. We are investigating the use of the FAP J10 VL for observation of septin proteins co-localization in the fungal pathogen Candida albicans. Because the universal CTG codon for leucine codes for serine in C. albicans, the FAP molecule of interest, in which 5 CTG codons have been identified, is affected. The purpose of this project is to make a J10 VL FAP tag that can be used in C.albicans. Using site directed mutagenesis and homologous recombination, CTG sites identified will be changed to TTG, a redundant codon for leucine. Gene sequencing will help determine successful mutagenesis and subsequent staining and imaging will help determine the utility of the FAP J10 VL– OTB bipartite fluoromolecule as a fluorescent tool for protein localization