Team:Cornell/project/wetlab/fungal toolkit/characterization
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Characterization
Flourescence
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has long been used as a molecular marker and reporter for eukaryotes and prokaryotes. Purified GFP absorbs blue light at a peak of 395 nm and emits green light at 509 nm, producing a stable fluorescence with little to no photobleaching [2]. GFP proved to be a valuable alternative to previous markers used in fungal research such as beta-glucuronidase, which was plagued with problems due to substrate uptake and leakiness [3]. Leaky gene expression is a result of initiation of transcription without the proper activator proteins. GFP was ideal because it does not require an exogenous substrate and does not negatively affect fungal tissue [3].In our studies, GFP and monomeric red fluorescent protein (mRFP) were utilized as markers for gene expression. In developing our fungal toolkit , we placed GFP and mRFP downstream of numerous promoters including the T7 promoter and fungal promoters PtrpC and A. nidulans PgpdA. The level of fluorescent activity would be indicative of promoter strength.
We are also collaborating with Wageningen's iGEM team to characterize their team's actin-GFP fusion construct in our chassis organisms.
References
[1] Biomol. (2004). Ampliqon III Competent Cells. Guide to Gene Expression in BL21, 1-12 Retrieved from http://www.biomol.de/dateien/infos_nr353.pdf[2] Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., & Prasher, DC. (1994). Science. Green Fluorescent Protein as a Marker for Gene Expression, 263. Retrieved from http://www.bio.davidson.edu/courses/molbio/restricted/02GFPwow/GFPwowpg1.html
[3] Maor, R., Puyesky, M., Horwitz, B. A., & Sharon, A. (1998). Mycology Research. Use of green fluorescent protein (GFP) for studying development and fungal-plant interaction in Cochliobolus heterostrophus, 102 (4), 491-496. Retrieved from http://www2.tau.ac.il/lifesci/plantsci/as/articles/chetgfp1.pdf
[4] Salis, H.M., Mirsky, E.A., & Voigt, C.A. (2010). Nat. Biotechnol. Automated Design of Synthetic Ribosome Binding Sites to Precisely Control Protein Expression, 27(10), 946-950. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2782888/pdf/nihms145791.pdf