Copy Number

Different cloning vectors are maintained at different copy numbers, dependent on the replicon of the plasmid (see Part 1). In a majority of cases in which a piece of DNA is cloned for maintenance and amplification for subsequent manipulation, the greater the yield of recombinant plasmid from E. coli cultures, the better. In this sce­nario, a high-copy-number vector is desirable such as those whose replication is driven by the ColEl replicon (8). The original ColEl-based plasmids have a copy number of 15-20. However, a mutant ColEl replicon, as found in the pUC series of plasmids (9), produces a copy number of 500-700 as a result of a point mutation within the RNAll regulatory molecule (see Part 1) that renders it more resistant to inhibition by RNAI (10). It should be noted that this mutation is temperature sensitive. Mutant RNAll is resistant to RNAl inhibition at 37°C or 42°C but not at 30°C, at which temperature the copy number of pUC plasmids returns to that of nonmutated ColE1 plasmids.

In some cases, a high-copy-number may cause problems for cloning DNA. For example, the cloned DNA may encode proteins that are toxic to the cell when present at high levels. This is particularly true of membrane proteins. Even if the protein is expressed poorly from the cloned DNA, the presence of many hundreds of copies of the gene on the plasmid may raise the level of protein to toxic levels. In these cases, using a plasmid with a lower copy number may reduce the gene dosage below a level at which toxicity occurs. For example, pBR322 is based on the original ColE1 replicon and thus has a copy number of 15-20 (11). The pACYC series of plasmids are based on the p15A replicon, which has a copy number of 18-22 (12). Low-copy-number plasmids include pSC101 (copy number around 5) (13), whereas BACs are maintained at one copy per cell (5).

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