Mutation in bacteria and their accompanying plasmids are observed to shape the fitness and functionality of the organism. While attempting to recombine a 125-bp insert into the Gateway® plasmid pDONR™P4-P1R, mutations were found within the attP4 integration site and ccdB gene. This led to the question of whether pDONR™P4-P1R is susceptible to increased spontaneous mutation under normal transformation conditions. Nine sources of pDONR™P4-P1R plasmids in the laboratory stocks under various storage conditions were each transformed sequentially for ten times and the attP4 site and a portion of the ccdB gene from the plasmid were sequenced from one transformant after each sequential transformation. Several common mutations were found within and near the ccdB gene of the plasmid, along with a rearrangement event that occurred in one of the plasmid generations. The measured mutation rate for this singular mutation was 1.3 X 10-5 per average number of nucleotides sequenced per sequential transformation. This is more than 2 X 104 times the rate of the predicted 5.4 X 10 -10 per nucleotide per replication for E. coli and for the F plasmid. These results suggest that pDONR™P4-P1R is susceptible to increased mutation under normal transformation conditions. Possible reasons for an increased rate of mutation are discussed.
|Advisor:||Vallier, Laura G., Krause, Maureen|
|School Location:||United States -- New York|
|Source:||MAI 51/02M(E), Masters Abstracts International|
|Subjects:||Molecular biology, Genetics|
|Keywords:||Attachment site, Mutation rate, PDONRP4 -P1R, Serial transformaion, Spontaneous mutation, ccdB gene|
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