A recombinase system facilitates cloning of expression cassettes in the ciliate -3

Copyright information:Taken from "A recombinase system facilitates cloning of expression cassettes in the ciliate "BMC Microbiology 2007;7():12-12.Published online 1 Mar 2007PMCID:PMC1839094.ce gene under the control of the promoter and the terminator (see figure 5B). It has a similar structure as the previously described cassette (resistance against paromomycin) that is also present in the here used acceptor plasmids pAX and pKOIX. Using the -dependent recombinase we generated the expression plasmids pAX-bsdR and pKOIX-bsdR and transformed strains. Blasticidin resistance assay: : clones of cells transformed with pAX-bsdR, clones of cells transformed with pKOIX-bsdR. Several independent clones (c1 to c10) were tested for bsd resistance. As controls we used the wildtype strain 1868/7 (WT) and a mock transformant (MT) that only carried the resistance gene growth control of clones in SPP-medium without antibiotics; same clones as presented in A selected in SPP-medium with 400 μg/mL paromomycin after 5–10 days same clones as in B, but cultivated for 3–5 days in SPP-medium with 100 μg/mL blasticidin; identical clones as in B/C cultivated in SPP-medium for 3–5 with both antibiotics, paromomycin (400 μg/mL) and blasticidin (100 μg/mL). strong growth of clones. cells alive, less growing. - no growth, cells died within 2–3 days.