PCR targeting system in Streptomyces coelicolor ... - Streptomyces UK

(E. coli Genetic Stock Center CGSC Strain # 7637) was modified by replacing the
ampicillin resistance gene bla with the chloramphenicol resistance gene cat,
generating pIJ790, to permit selection in the presence of Supercos1-derived cosmids
(ampicillin and kanamycin resistance).
Name of
plasmid
pIJ773
Fig. 5
pIJ778
Fig. 6
Resistancemarker
Resistance
Concentration
for E. coli
oriT Size of template
aac(3)IV apramycin 50 μg/ml LB + 1382 bp
aadA
pIJ779. aadA
pIJ780
Fig.7
spectinomycin
streptomycin
spectinomycinstreptomycin
50 μg/ml LB
50 μg/m LB
50 μg/ml LB
50 μg/ml LB
+ 1425 bp
- 1057 bp
vph viomycin 30 μg/ml DNA + 1497 bp
pIJ781 vph viomycin 30 μg/ml DNA - 1622 bp
Table 1: Disruption cassettes containing different resistance markers with and without oriT: All
disruption cassettes were cloned into the EcoRV site of pBluescript SK II (+) allowing the isolation of
a EcoRI/HindIII fragment for use as template for the PCR reaction. The size of the cassettes includes
the 19 bp and 20 bp primer site (see section 2: “primer design”) which are identical in all disruption
cassettes. The resistance genes with or without oriT are flanked by FRT sites (FLP recognition targets)
which allows FLP-mediated excision of the cassette (see section 7: “FLP-mediated excision of the
disruption cassette”).
2