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Fig. 3 | Biotechnology for Biofuels

Fig. 3

From: Mutant generation by allelic exchange and genome resequencing of the biobutanol organism Clostridium acetobutylicum ATCC 824

Fig. 3

Screening of Spo0A mutants and ACE corrected/complemented/overexpressed clones. Key MW, 2-log DNA marker (NEB) molecular weight marker; ‘W’, is a PCR with no DNA; ‘P’, is a PCR using the plasmid only, and; ‘E’ is the pyrE minus parent strain CRG1545. On b, c the final arrangement of the chromosomal region in the pyrE repaired strains is illustrated. Components are: dcd, deoxycytidine triphosphate deaminase; cac0026, hypothetical protein; pyrE, orotate phosphoribosyltranspherase; Pspo0A, promoter of the spo0A gene; Pfdx, promoter of the C. pasteurianum ferredoxin gene; spo0A, encodes the master regulator of sporulation, Spo0A; lacZ’, alpha-peptide of β-galactosidase gene containing multiple cloning sites; TT, transcriptional terminator found between hydA (hydrogenase) and pyrE. Arrows above and below the sequence labelled 1 and 2 show position of primers specific to each experiment. Lanes 19 (a), 12 (b) and 18 (c, d) are the screened DNA samples from randomly selected uracil prototrophic clones. a PCR screening of nine FOAR colonies using flanking primers Cac-spo0A-sF2 (1) and Cac-spo0A-sR2 (2). The expected PCR product for the mutant is 1679 bp, for the parent strain CRG1545 is 2114 bp. b PCR screening of the two uracil prototroph clones using flanking primers Cac0026-sF2 (1) and Cac-hydA-sR2 (2). The expected PCR product for the pyrE mutant parent strain is 1936 bp and for the strain in which the pyrE allele has been restored to wild-type with pMTL-ME6 is 1989 bp. c PCR screening of eight uracil prototroph using flanking primers Cac0026-sF2 (1) and Cac-hydA-sR2 (2). The expected PCR product for the parent pyrE minus strain is 1936 bp and for the strain in which the pyrE allele has been restored to wild-type with pMTL-ME6C::spo0A is 3610 bp. d PCR screening of eight uracil prototroph using flanking primers Cac0026-sF2 (1) and Cac-hydA-sR2 (2). The expected PCR product for the pyrE mutant parent strain is 1936 bp and for the strain in which the pyrE allele has been restored to wild-type with pMTL-ME6X::spo0A is 3450 bp

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