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. 2019 Feb 18;4(1):bpz001.
doi: 10.1093/biomethods/bpz001. eCollection 2019.

Genetic transformation of Coniochaeta sp. 2T2.1, key fungal member of a lignocellulose-degrading microbial consortium

Nancy N Nichols  1 , Ronald E Hector  1 , Sarah E Frazer  1
Affiliations

Genetic transformation of Coniochaeta sp. 2T2.1, key fungal member of a lignocellulose-degrading microbial consortium

Nancy N Nichols et al. Biol Methods Protoc. .

Abstract

Coniochaeta sp. strain 2T2.1 is a key member of a microbial consortium that degrades lignocellulosic biomass. Due to its ecological niche and ability to also grow in pure culture on wheat straw, protocols for transformation and antibiotic selection of the strain were established. Hygromycin was found to be a reliable selectable transformation marker, and the mammalian codon-optimized green fluorescent protein was expressed and used to visualize fluorescence in transformed cells of strain 2T2.1.

Keywords: GFP; biomass; fungi; protoplast; transformation.

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Figures

Figure 1:
Figure 1:
Brightfield microscopic image of C. sp. 2T2.1 growing in association with ground wheat straw.
Figure 2:
Figure 2:
Effect of lysing enzymes (A) concentration and (B) incubation time on C. sp. 2T2.1 transformation efficiency. (A) Varied concentrations of lysing enzymes were incubated for 60 min; (B) lysing enzymes (80 mg/ml) were incubated for varying times during protoplast formation. Error bars indicate standard deviation of three to five replicates.
Figure 3:
Figure 3:
C. sp. 2T2.1 transformed with pJQ7. (A) Fluorescence image of cells grown in YPD medium; (B) same field as A, with green outlines showing cells counted as fluorescent and red outlines showing nonfluorescing cells; (C) brightfield image of cells grown in liquid mineral medium containing ground wheat straw as carbon source and (D) same field as C; fluorescence image.
Figure 4:
Figure 4:
Stability of fluorescence in a C. sp. 2T2.1 pJQ7 transformant. Median fluorescence is shown for cells cultured with hygromycin (black bars) or without hygromycin (gray bars). Error bars indicate standard deviation for four replicates in subcultures 1 through 4 and two replicates in subcultures 5 and 6.
Figure 5:
Figure 5:
Variation in fluorescence intensity among C. sp. strain 2T2.1 transformants expressing GFP. Individual transformed colonies were cultured in YPD containing hygromycin for measurement. Bars indicate standard deviation of three cultures for each transformant. Asteriks indicate untransformed negative control.
See this image and copyright information in PMC

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