Abstract
Genetic engineering of fungal strains is highly important for meeting the requirements of tailor-made applications in biotechnology. Aiming targeted strain design we describe a transformation approach for filamentous fungi that favours homologous integration and permits multiple rounds of gene deletions by applying a reusable and bidirectionally functional marker system.
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Notes
- 1.
This reagent is used for growth restriction in order to obtain clearly confined colonies from streaked spores.
- 2.
For the PCR reactions some optimization may be required according to particular primer sets. The reactions are normally carried out in 50-μL mixtures containing 1.25 U GoTaq Flexi DNA polymerase (Promega), 2 mM MgCl2, 1X Green GoTaq Flexi buffer, 0.2 mM dNTPs, 0.1 μM of forward and reverse primer each, 1 μL of template DNA (10 ng plasmid DNA, 300 ng genomic DNA), and nuclease-free water. The standard PCR program consists of an initial denaturation of 3 min at 95 °C, followed by 35 cycles each comprising 30 s at 95 °C, 30 s at 60 °C, and 1 min/kb at 72 °C.
- 3.
For efficient gene replacement events in Trichoderma, it is necessary to produce cassettes with long (~1,000 bp) 5′- and 3′-regions flanking the desired integration locus (Guangtao et al. 2009). Because of this high minimum length of the flanking regions, it may be difficult to find suitable restriction enzymes for the cloning procedure of the deletion cassette. The usage of the Cre recombinase reaction is an alternative to overcome this problem. Nevertheless, classical cloning techniques may still be employed to create the desired version of pMS-5loxP3 depending on the sequences of the flanking regions of the target gene. The whole fragment bearing the two loxP-sites and the amdS and hph genes (5,375 bp) can be excised from pMS-HALS by using SalI and NotI restriction sites (Fig. 14.1).
- 4.
Please note that the pki::hph expression cassette, which is present in pMS-HALS, is fully functional in E. coli. This allows the screening of positive clones from the E. coli transformation by selection on hygromycin B and enables the use of this plasmid as shuttle system between E. coli and the fungus. Consequently, the donor vector pMS-HALS is generally highly useful for the construction of any deletion cassette.
- 5.
If E.coli supercharge EZ10 electrocompetent cells are not available, other strains such DH5α or XL1-Blue can be successfully used only resulting in differences of the transformation efficiency.
- 6.
Performing one round of marker re-use did not result in unwanted recombination events of the present loxP-site and the new loxP-sites (Steiger et al. 2011). However, after having performed all desired rounds of gene deletions, the elimination of the Cre recombinase is strongly recommended to construct a genetically stable strain with respect to its loxP-sites. In QM6aΔtmus53Δpyr4 the cre gene was directed to the pyr4 locus. In a final transformation step the pyr4 gene is used to replace the cre gene, thereby employing the pyr4 gene as a marker. The resulting, positively transformed strain gains uridine prototrophy and does not contain the Cre recombinase anymore. This has been demonstrated by the successful pyr4 retransformation into strain QM6aΔtmus53Δpyr4, which generated QM6aΔtmus53. The latter is still NHEJ-deficient and can be employed for gene replacement aiming homologous integration (Steiger et al. 2011).
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Acknowledgments
This work was supported by three grants from the Austrian Science Fund (FWF): [P21287, V232-B20, P24851] given to RLM and ARMA, respectively, and by a doctoral programme of Vienna University of Technology (CatMat).
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Mello-de-Sousa, T.M., Mach, R.L., Mach-Aigner, A.R. (2015). A Recyclable and Bidirectionally Selectable Marker System for Transformation of Trichoderma . In: van den Berg, M., Maruthachalam, K. (eds) Genetic Transformation Systems in Fungi, Volume 2. Fungal Biology. Springer, Cham. https://doi.org/10.1007/978-3-319-10503-1_14
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