Plasmid backbones/Inducible copy number/Features

Sometimes it is nice to have the best of both worlds. For example, you'd usually like a BioBrick® part to propagate at low copy, but you'd also like to switch the part over to high copy when you're interested in purifying plasmid DNA. The solution is an inducible copy number plasmid backbone. Both plasmid backbones listed below have a high copy replication origin under the control of a LacI-repressible promoter. When the plasmid is propagated in an E. coli strain that expresses the repressor lacI in high quantities, such as D1210 (or BBa_V1003), the plasmid is maintained at low copy by the F' origin. To increase the plasmid copy number, simply add the small molecule [http://openwetware.org/wiki/IPTG IPTG] to induce the LacI-repressible promoter controlling the high copy number origin.

  • Note: because these inducible copy number vectors rely on LacI for control of the high copy number origin, they are incompatible with any BioBrick® part, device, or system that include lacI or a LacI-regulatable promoter.

The plasmid backbones listed below have a common set of features.

  1. A complete BioBrick® cloning site for easy cloning and assembly of BioBrick parts.
  2. Terminators flanking the BioBrick® cloning site to insulate the vector from read-through transcription originating in the cloned BioBrick® part, device or system.
  3. Primer binding sites for the standard BioBrick® verification primers VF2 (BBa_G00100) and VR (BBa_G00101). These primers are located for convenient sequencing and [http://openwetware.org/wiki/Colony_PCR screening by colony PCR] of cloned BioBrick® parts, devices, and systems.

Plasmid backbones are distributed by the Registry with a default insert. There are just a handful of default plasmid inserts used in the Registry. Many the available plasmid backbones have the ccdB positive selection marker (BBa_P1010) as the default plasmid insert within the BioBrick® cloning site. The ccdB gene ensures that when assembling two BioBrick® parts together, the uncut plasmid is not transformed. However, inclusion of the ccdB gene means that these vectors must be propagated in a ccdB tolerant strain, such as E. coli strain DB3.1 (BBa_V1005).