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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

A95 transformants. A- three weeks after PEG mediated transformation of A95 protoplasts with pCB1004, single A95 colonies were observed on malt-extract agar plates containing hygromycin. B- PCR products of the hph-gene (1,019 bp) seen on agarose gels. Polymerase chain reaction detection of the hph-gene from DNA of five independent putative transformants confirmed integration of hph into the genome of A95 transformants. Arrow indicates location of hph DNA bands.
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Figure 3: A95 transformants. A- three weeks after PEG mediated transformation of A95 protoplasts with pCB1004, single A95 colonies were observed on malt-extract agar plates containing hygromycin. B- PCR products of the hph-gene (1,019 bp) seen on agarose gels. Polymerase chain reaction detection of the hph-gene from DNA of five independent putative transformants confirmed integration of hph into the genome of A95 transformants. Arrow indicates location of hph DNA bands.

Mentions: Colonies were visible on hygromycin B containing MEAS agar two to three weeks after transformation of A95 protoplasts with pCB1004 (Figure 3A). Five randomly selected, transformed colonies were able to grow when plated out again on hygromycinB-containing agar. Total DNA from some of these re-picked colonies was amplified by PCR using the primer pair hphRforw/hphRrev resulting in an amplicon of ≈ 1,000 bp, which is consistent with the size (1,020 bp) of the hph-gene (Figure 3B). The sequences of all PCR products matched completely those of hph. About 55 transformants were obtained from 106 protoplasts in a total volume of 150 μl containing 10 to 20 μg plasmid DNA.


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)

A95 transformants. A- three weeks after PEG mediated transformation of A95 protoplasts with pCB1004, single A95 colonies were observed on malt-extract agar plates containing hygromycin. B- PCR products of the hph-gene (1,019 bp) seen on agarose gels. Polymerase chain reaction detection of the hph-gene from DNA of five independent putative transformants confirmed integration of hph into the genome of A95 transformants. Arrow indicates location of hph DNA bands.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: A95 transformants. A- three weeks after PEG mediated transformation of A95 protoplasts with pCB1004, single A95 colonies were observed on malt-extract agar plates containing hygromycin. B- PCR products of the hph-gene (1,019 bp) seen on agarose gels. Polymerase chain reaction detection of the hph-gene from DNA of five independent putative transformants confirmed integration of hph into the genome of A95 transformants. Arrow indicates location of hph DNA bands.
Mentions: Colonies were visible on hygromycin B containing MEAS agar two to three weeks after transformation of A95 protoplasts with pCB1004 (Figure 3A). Five randomly selected, transformed colonies were able to grow when plated out again on hygromycinB-containing agar. Total DNA from some of these re-picked colonies was amplified by PCR using the primer pair hphRforw/hphRrev resulting in an amplicon of ≈ 1,000 bp, which is consistent with the size (1,020 bp) of the hph-gene (Figure 3B). The sequences of all PCR products matched completely those of hph. About 55 transformants were obtained from 106 protoplasts in a total volume of 150 μl containing 10 to 20 μg plasmid DNA.

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