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Genetic transformation of Knufia petricola A95 - a model organism for biofilm-material interactions.

Noack-Schönmann S, Bus T, Banasiak R, Knabe N, Broughton WJ, Den Dulk-Ras H, Hooykaas PJ, Gorbushina AA - AMB Express (2014)

Bottom Line: This mixture was equally effective on the melanin-minus mutant and the type-strain.Stability of transformation was confirmed by sub-culturing the putative transformants on selective agar containing HmB as well as by PCR-detection of the hph gene in the colonies.The hph gene was stably integrated as shown by five subsequent passages with and without selection pressure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department 4 (Materials & Environment), Federal Institute for Materials Research and Testing (Bundesanstalt für Material-forschung und -prüfung, BAM), Unter den Eichen 87, Berlin, 12205, Germany.

ABSTRACT
We established a protoplast-based system to transfer DNA to Knufia petricola strain A95, a melanised rock-inhabiting microcolonial fungus that is also a component of a model sub-aerial biofilm (SAB) system. To test whether the desiccation resistant, highly melanised cell walls would hinder protoplast formation, we treated a melanin-minus mutant of A95 as well as the type-strain with a variety of cell-degrading enzymes. Of the different enzymes tested, lysing enzymes from Trichoderma harzianum were most effective in producing protoplasts. This mixture was equally effective on the melanin-minus mutant and the type-strain. Protoplasts produced using lysing enzymes were mixed with polyethyleneglycol (PEG) and plasmid pCB1004 which contains the hygromycin B (HmB) phosphotransferase (hph) gene under the control of the Aspergillus nidulans trpC. Integration and expression of hph into the A95 genome conferred hygromycin resistance upon the transformants. Two weeks after plating out on selective agar containing HmB, the protoplasts developed cell-walls and formed colonies. Transformation frequencies were in the range 36 to 87 transformants per 10 μg of vector DNA and 10(6) protoplasts. Stability of transformation was confirmed by sub-culturing the putative transformants on selective agar containing HmB as well as by PCR-detection of the hph gene in the colonies. The hph gene was stably integrated as shown by five subsequent passages with and without selection pressure.

No MeSH data available.


Related in: MedlinePlus

Plasmid map of pCB1004. Plasmid pCB1004 was constructed by Carroll et al. ([1994]). The vector has a chloramphenicol resistance- and a functional lacZ-gene for blue-white screening. hph = Hygromycin B resistance; trpC = A. nidulands promoter.
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Figure 2: Plasmid map of pCB1004. Plasmid pCB1004 was constructed by Carroll et al. ([1994]). The vector has a chloramphenicol resistance- and a functional lacZ-gene for blue-white screening. hph = Hygromycin B resistance; trpC = A. nidulands promoter.

Mentions: The hygromycin selection vector for PEG mediated transformation pCB1004 (Carroll et al. [1994]) was used. pCB1004 contains the hygromycin B phosphotransferase gene (hph) under control of the A. nidulans trpC promoter (Mullaney et al. [1985]) but without the trpC terminator. pCB1004 also contains a chloramphenicol resistance cassette, a functional lacZ gene and a multiple cloning site (Figure 2).


Genetic transformation of Knufia petricola A95 - a model organism for biofilm-material interactions.

Noack-Schönmann S, Bus T, Banasiak R, Knabe N, Broughton WJ, Den Dulk-Ras H, Hooykaas PJ, Gorbushina AA - AMB Express (2014)

Plasmid map of pCB1004. Plasmid pCB1004 was constructed by Carroll et al. ([1994]). The vector has a chloramphenicol resistance- and a functional lacZ-gene for blue-white screening. hph = Hygromycin B resistance; trpC = A. nidulands promoter.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230810&req=5

Figure 2: Plasmid map of pCB1004. Plasmid pCB1004 was constructed by Carroll et al. ([1994]). The vector has a chloramphenicol resistance- and a functional lacZ-gene for blue-white screening. hph = Hygromycin B resistance; trpC = A. nidulands promoter.
Mentions: The hygromycin selection vector for PEG mediated transformation pCB1004 (Carroll et al. [1994]) was used. pCB1004 contains the hygromycin B phosphotransferase gene (hph) under control of the A. nidulans trpC promoter (Mullaney et al. [1985]) but without the trpC terminator. pCB1004 also contains a chloramphenicol resistance cassette, a functional lacZ gene and a multiple cloning site (Figure 2).

Bottom Line: This mixture was equally effective on the melanin-minus mutant and the type-strain.Stability of transformation was confirmed by sub-culturing the putative transformants on selective agar containing HmB as well as by PCR-detection of the hph gene in the colonies.The hph gene was stably integrated as shown by five subsequent passages with and without selection pressure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department 4 (Materials & Environment), Federal Institute for Materials Research and Testing (Bundesanstalt für Material-forschung und -prüfung, BAM), Unter den Eichen 87, Berlin, 12205, Germany.

ABSTRACT
We established a protoplast-based system to transfer DNA to Knufia petricola strain A95, a melanised rock-inhabiting microcolonial fungus that is also a component of a model sub-aerial biofilm (SAB) system. To test whether the desiccation resistant, highly melanised cell walls would hinder protoplast formation, we treated a melanin-minus mutant of A95 as well as the type-strain with a variety of cell-degrading enzymes. Of the different enzymes tested, lysing enzymes from Trichoderma harzianum were most effective in producing protoplasts. This mixture was equally effective on the melanin-minus mutant and the type-strain. Protoplasts produced using lysing enzymes were mixed with polyethyleneglycol (PEG) and plasmid pCB1004 which contains the hygromycin B (HmB) phosphotransferase (hph) gene under the control of the Aspergillus nidulans trpC. Integration and expression of hph into the A95 genome conferred hygromycin resistance upon the transformants. Two weeks after plating out on selective agar containing HmB, the protoplasts developed cell-walls and formed colonies. Transformation frequencies were in the range 36 to 87 transformants per 10 μg of vector DNA and 10(6) protoplasts. Stability of transformation was confirmed by sub-culturing the putative transformants on selective agar containing HmB as well as by PCR-detection of the hph gene in the colonies. The hph gene was stably integrated as shown by five subsequent passages with and without selection pressure.

No MeSH data available.


Related in: MedlinePlus