MA317 transformants containing the
using the pyrG**
targeting method. A) Southern blot analysis. Genomic DNA was restricted with EcoRI or KpnI and size fractioned by electrophoresis on a 1.0 % agarose gel. For hybridisation, 32P-labelled pyrG probe (1255 bp, Figure 2A) or 3’ pyrG probe (684 bp, Figure 2A) were used. When digested with EcoRI and using the pyrG probe (upper panel), a signal of 9.0 kb corresponds with the wild-type pyrG locus, while a signal of 4.9 kb corresponds with integration of the Pgpd-mluc cassette at the pyrG locus. When digested with KpnI and using the 3’ pyrG probe (lower panel), a signal of 3.3 kb corresponds with the wild-type pyrG locus, while a signal of 4.8 kb corresponds with integration of the Pgpd-mluc cassette at the pyrG locus. Strains MA317.1 and MA317.3 have the wild-type pyrG locus, while strains MA317.2 and MA317.4-6 contain the Pgpd-mluc cassette at the pyrG locus. B) Lux activity assay. The lux activity assay described by Meyer et al.  has been slightly modified. Briefly, 100 μL of 2 x Minimal Medium  with 0.006 % yeast extract (w/v), 76 μL deionized water, 4 μL 25 mM luciferin (Promega, E1605) and 20 μL spore suspension (7.5*105 spores/mL) was pipetted together (in triplicate) in a well of a white, clear bottom, 96 wells plate (Greiner Bio-one, ref 655095) and incubated for 24 hours at 30 °C in the EnSpire Multiplate Reader (Perkin) with continuous measuring of lux and OD. Lux activities after 16 h of incubation are shown here. Strains MA317.1 and MA317.3 have no lux activity, while strains MA317.2 and MA317.4-6 show comparable lux activities.