Vary, Patricia S.
B.S. (Bachelor of Science)
Department of Biological Sciences
in the Hind HI site of ORF A. Since pKM3 had two Hind HI sites (one is in the multiple cloning site of the vector pJM103 and the other is in ORF A) the ORF A fragment was first transferred into vector pGEM5Z which does not have any Hind in sites and then the kanamycin cassette was inserted. Protoplast transformation into B. megaterium PV361 yielded no kanamycin resistant colonies and controls were positive in three experiments, therefore, it can be concluded that ORF A was inactivated. To test whether the inactivated ORF A could then be complemented, PV361 containing a plasmid with a functional ORF A was transformed with the defective clone. Surprisingly, there were no colonies on the plate, indicating that there was no complementation. This experiment was repeated four times with no complementation. The sequence of ORF A was rechecked and it was discovered that the kanamycin cassette has been placed between the direct repeats, which is the presumed binding site for the replication protein, and the GC and AT rich regions which are also necessary for replication. Hence, insertion of the kanamycin cassette not only inactivated the replication protein, but also the origin sequence. Therefore, the defective ORF A clone was not able to be complemented by the functional ORF A. Currently we are trying to inactivate ORF A using PCR to delete the 5' amino terminal end and the 3' carboxy terminal end without disturbing the bining site.
Marszalik, Julie A., "Determining the replication protein for the 71 kilobase plasmid of Bacillus megaterium QM B1551." (1997). Honors Capstones. 400.
13 pages, 9 unnumbered pages
Northern Illinois University
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