Table 2. Excision of skinCd during growth in strains producing CD1234 under Ptet control.
| ΔCt = CtsigK-CtpolIII | Detection of skinCd junction by PCR | ||
|---|---|---|---|
| Strain | pDIA6103 | pDIA6103-Ptet-CD1234 | |
| 630Δerm | 10.9+/-0.5 | 0.8+/-0.4 | 0/8 |
| sigE::erm | 17.9+/-0.5 | 0.6+/-0.5 | 1/8 |
| spoIIID::erm | 16.3+/-0.15 | 0.85+/-0.35 | 1/8 |
| CD1231::erm | 18.3+/-0.25 | 18+/-1 | 8/8 |
| CD1234::erm | 17.9+/-0.25 | 0.65+/-0.35 | 1/8 |
Strains 630Δerm, sigE::erm, spoIIID::erm, CD1234::erm and CD1231::erm containing either pDIA6103 or pDIA6103-Ptet-CD1234 were grown in TY medium for 4 h at which time ATc (100 ng/ml) was added. After 2 h of induction, cells were serially diluted and plated on BHI or collected and DNA extracted. qPCR was performed on with 2 primer pairs: one corresponding to DNApolIII as a control and the second to sigK on both sides of the skin insertion site (qRTBD325-qRTBD326). Chromosomal DNA extracted from 8 independent clones obtained after plating the cultures on BHI was used to amplify the 5’ junction of the skin using one primer in sigK (qRTBD326) and one in CD1231 (OBD742) (See Fig 6A). The number of positive clones among the eight tested is indicated.