School of Engineering and Science - Jacobs University

Results
If integration is due to a homologues double cross over event, the plasmid
backbone is lost. Thus the Pss22d transformants were screened for kanamycin
resistance and the resulting candidates were than screened for a lack of
ampicillin resistance. Finally, integration of the marker exchange cassette by
double crossover was verified by Southern blot analysis (Fig.8). Plasmid
pPVSA4 (control) and genomic DNA of wild type (wt) Pss22d and of 5 possible
transformants were digested by ApaLI, separated by agarose gelelectrophoresis,
transferred to a membrane and then hybridized at 60°C. Three
separate blots were analyzed by hybridization with probes against the
pGEM-T Easy vector backbone, the kanamycin cassette, and pvsA PCR
fragment1. Transformant no. 5 showed the expected hybridization pattern for
double cross-over insertion: There is no signal for the vector backbone, but
there is a signal for the kanamycin cassette.
probe:
vector backbone kanamycin cassette
pvsA fragment 1
K M wt 1 2 3 4 5 K M wt 1 2 3 4 5 K M wt 1 2 3 4 5
8
5
4
3
2
1,6
size (kb)
Fig. 8. Verification of the Pss22d∆Pvd genotype by Southern blot analysis.
Five different transconjugants were tested for correct insertion of the marker-exchange cassette by
double-homologues recombination. Sample DNA was digested with ApaL1 and loaded as follows:
K= control plasmid pPVSA4; M= 1kb molecular weight standard; wt= Pss22d; 1-5 = Transformants
of Pss22d electroporated with pPVSA4 and screened for kanamycin resistance. Three separate
blots were analysed by hybridisation with probes against the pGEM-T Easy®-vector backbone, the
kanamycin cassette, and pvsA PCR fragment 1, respectively. The red boxes indicate the
hybridisation pattern of Pss22d wild type and the double cross-over mutant no. 5.
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