Looking for low copy number plasmids

Overview and Approach

Our team faced several problems in finding different copy number plasmids in the list of iGEM standard backbones available in the registry. In total the iGEM registry has eight different Origens of replication (Ori) documented, numbered from pSB1 to pSB8, having different copy numbers. For our current interest, pSB1 (Ori modified pMB1 derived from pUC19)is the only documented high copy number plasmid, pSB3 (p15A Ori, pSB4 (rep101, repA Ori) and pSB6 (pMB1 Ori)are described as low-medium copy number plasmids. At the beginning of the project we used pS1K3 and pSB6A3, and by our surprise the obtained concentrations were in the same range, when we were expected different concentrations. This is the main reason why we decided to compare the copy number of the different plasmids by growing 10 mL E. coli in LB medium at an Optical Density (OD) of 0.6, minipreping it and determine its concentration, as well as, running the samples in an agarose gel. All these was done in triplicates, except for the high copy number plasmid pSB1K3 that was preformed in duplicates.

Results

The performed experiments show that pSB6 has a higher copy number plasmid than pSB1, the iGEM standard high copy number plasmid. Importantly pSB3 is the only real low copy number plasmid in comparison to the other backbones.

These findings are relevant for iGEM teams from the first day when they will plan their experiments based on the information found in the registy.