If I want to construct a plasmid containing only the E. coli chromosomal origin of replication fragment and the pUC19 backbone with its antibiotic resistance, how should I proceed?
I could first design two pairs of PCR primers to amplify the target fragments. Subsequently, perform restriction enzyme digestion, ligate the fragments using T4 DNA ligase, transform the ligation product into competent cells, and screen for transformants using antibiotic selection. Positive clones would then be picked for sequencing verification.
The origin of replication in my constructed plasmid is identical to that of the chromosomal origin in the competent host cells. Could this identity interfere with the replication of my plasmid?
First of all, you should know this has been done many times and the E. coli oriC has been well studied. You might want to review the literature first of all to be sure you know how large a fragment you need to get from the chromosome.
Secondly, are you cloning it into a plasmid with its own pUC19 origin also, or just the E. coli origin? If the latter then the copy number will be 1 just like the E. coli origin. If the plasmid origin is still present then it will have the plasmid copy number.
I don't think you will see any interference but again this is all known and is in the literature.
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