Abstract
We present a collection of minimalist binary vectors for transformation through ATMT applicable to several fungi species. pLUO plasmid binary vectors consist of a reporter module containing fluorescent proteins, mCherry or eGFP, flanked by a multiple cloning site and a transcription terminator site. They also present a synthetic gene allowing resistance to Hygromicin B flanked by alternate promoters, one for yeast and another for filamentous fungi. Left and right borders were added for Agrobacterium tumefaciens recognition, and a minimal broad-host range RK2 replication origin. Transformation was validated in the pathogenic fungus Paracoccidioides lutzii. Hence, we developed an efficient and reliable molecular tool for fungal transformation: minimalist, synthetic, modular, and available in four different versions, and these can still be readily modified using a few primers and few cloning steps.
Highlights
Fungi are organisms comprising a universe that has not been fully explored by mankind(Leigh et al, 2003), but have been extensively studied because of their huge impact in everyday life and their endless applications in industry, such as production of biofuels (Glass et al, 2013), foods and feedstock (Bhat, 2000), human therapeutics (Ward, 2012), among many others
In the pursuit of overcoming the lack of tools for fungal studies, we developed the pLUO vectors, a collection of minimal and versatile binary plasmid vectors for A. tumefaciens-mediated transformation (ATMT)
This was achieved by employing the pGLR2 plasmid as vector backbone (Benedetti et al, 2012) that is minimum and presents a broad host range RK2 origin of replication, so it replicates in E. coli and in A. tumefaciens. pLUO vectors present a multiple cloning site (MCS) with 11 different restriction sites for several cloning options, so any given promoter can be placed to modulate a red or a green reporter protein
Summary
Fungi are organisms comprising a universe that has not been fully explored by mankind(Leigh et al, 2003), but have been extensively studied because of their huge impact in everyday life and their endless applications in industry, such as production of biofuels (Glass et al, 2013), foods and feedstock (Bhat, 2000), human therapeutics (Ward, 2012), among many others. In the pursuit of overcoming the lack of tools for fungal studies, we developed the pLUO vectors, a collection of minimal and versatile binary plasmid vectors for A. tumefaciens-mediated transformation (ATMT). PLUO vectors present a multiple cloning site (MCS) with 11 different restriction sites for several cloning options, so any given promoter can be placed to modulate a red (mCherry) or a green (eGFP) reporter protein.
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