SUPPLEMENTAL FIGURE S1 Cloning strategy of the Tet-On expression system. Construction of pVG1-47 (P gpdA:: rtTA::T cgrA – TetO7:: P min:: T trpC )
Cloning strategy of the Tet-On expression system
Construction of pVG1-47 (PgpdA::rtTA::TcgrA –TetO7::Pmin::TtrpC)
A 520 bp TetO7-Pmin fragment and a 436 bp TtrpC fragment were PCR amplified from plasmid p500 (Vogt et al., BMC Microbiol 5:1.,2005) by introducing the indicated restriction site overhangs. The PCR product obtained were cloned into pJET (Fermentas) giving plasmids pSA2-5 and pSA3-6, respectively. Both the TetO7-Pmin fragment and the TtrpC fragment were cut out from pSA2-5 and pSA3-6 via a NotI x PmeI double restriction and cloned into the NotI site of p474 (Vogt et al. , BMC Microbiol 5:1.,2005) via a three-way-ligation.
Construction of pVG2.2 (PgpdA::rtTA::TcgrA– TetO7:: Pmin::TtrpC– pyrG*)
The 2.2 kb pyrG* fragment was PCR amplified from pAB94 (van Gorcom and van den Hondel, Nucleic Acids Res 16:9052, 1988) by introducing AscI overhangs and cloned into the AscI site of pVG1-47.
Construction of pVG3 (PgpdA::rtTA::TcgrA – TetO7::Pmin::mluc::TtrpC)
The 1.6 kb mluc open reading fragment was PCR amplified from pLUC6 (Morgan et al., Fungal Genet Biol 38:327-32, 2003) by introducing PmeI overhangs and cloned into pJET vector (Fermentas) yielding in plasmid pSA1-2. The mluc fragment was cut from pSA1-2 via PmeI restriction and cloned into the PmeI site of pVG1.
Construction of pVG4.1 (PgpdA::rtTA::TcgrA – TetO7::Pmin::mluc::TtrpC – pyrG*)
The pyrG* AscI-fragment (see above) was ligated into the AscI site of pVG3, creating pVG4.1.
Color code: Green (A) = PgpdA::rtTA::TcgrA, purple (B) = tetO7::Pmin::TtrpC, red (C) = pyrG* and orange = mluc. The sizes of the fragments and the plasmids are indicated.
Southern analysis of A. niger strains gained after transformation with pVG2.2 or pVG4.1
To confirm homologous integration of the constructs at the A. nigerpyrG locus, genomic DNAs of selected transformants were restricted with NcoI and subjected to Southern hybridization using pyrG as a probe. Strain AB4.1 served as a wild type control. NcoI does not cut within pVG2.2 but once within the mluc fragment of pVG4.1.
The expected fragment size for the wild type pyrG is 3.2 kb. For a single-copy integration of construct pVG4.1 at pyrG, two signals are expected (5 kb, 7.7 Kb). For a single-copy integration of construct pVG2.2 at pyrG, one signal at 11.8 Kb is expected. For a tandem-copy integration of construct pVG4.1 at pyrG, three signals are expected (5 kb, 7.7 kb, 9.4 kb).
Lanes 1 and 15: control strain AB4.1 showing a single band of 3.2 kb
Lane 3: strain VG5.1, single-copy integration of pVG2.2 at pyrG (one signal at 11.8 Kb)
Lane 6: heterokaryon, single-copy integration of pVG4.1 at pyrG (two signals at 5 and 7.7 kb plus a wild type signal at 3.2 kb)
Lane 7: strain VG6.1, single-copy integration of pVG4.1 at pyrG (two signals at 5 and 7.7 kb)
Lane 10: strain VG8.1, integration of two copies of pVG4.1 at pyrG resulting in three signals (5, 7.7, 9.44 kb)
Lane 11: strain VG8.2, integration of two copies of pVG4.1 at pyrG resulting in three signals (5, 7.7, 9.44 kb) & heterologous integration of one pVG4.1 copy (signal > 11 kb)
Lanes 2, 4, 5, 8, 9, 12-14 show wild type signal, indicating restoration of a functional pyrG allele without plasmid integration.