Synthetic Biology:Vectors/Single copy plasmid

See Vectors for information on vector nomenclature, existing vectors and vectors that we would like constructed.

Construction of single copy BioBricks vector

See notes on bacterial artificial chromosomes.

Proposed features

• F plasmid backbone
• positive selection marker (i.e. ccdB or sacB) in between BioBricks restrictions sites to facilitate cloning
• some mechanism for putting the vector at high copy for purification purposes
• strong terminators flanking the BioBricks insertion site
• no loxP or cos insertion sites or Tn7 attachment sites?
  • I can't think of an obvious use of these sites unless we want to build in the capability for integrating onto the genome. However, wouldn't it make more sense to build in recombination capabilities onto a higher copy number vector than this?
• no blue-white screening?
  • inclusion of a lacZα fragment would restrict its use as a part
• multiple versions with different antibiotic resistance markers
• no selection system for mammalian cells
• VF2 and VR sites

Drawbacks

• Can only be used in F^- strains
• Should likely be used in recA^- strains to avoid integration onto the genome and ensure plasmid stability.
• It is unclear whether this vector would truly be operating at single copy. If it is not, perhaps it is easier to stick with the pSB2* plasmids.

To do list

• One of the things need for this project is BioBricked antibiotic resistance cassettes. This is also a prerequisite for the standard strain project. Tom has ordered primers and is planning on cloning several resistance cassettes.
• Reshma: specify F plasmid backbone with no internal BioBricks restriction sites
  • Ask Randy to get it synthesized?
• ccdB is already in BioBricks form
• Plan assembly of vector modules.
• Identify strong terminators to flank the BioBricks insertion site

To be decided

• If all the vector components are specified in BioBricks format, how do we construct a BioBricks insertion site?
  • Blunt-end ligation?
  • Other restriction enzyme sites?
  • PCR
• Use an secondary, inducible copy number origin or insert the pUC backbone inside of the BioBricks cloning site?
  • Inducible copy number origin
    • The most common system for inducible copy number F-based plasmid requires a special strain (a copy up mutant of traF under the control of an arabinose inducible promoter) for inducible behavior.
    • The plasmid should operate at single copy in most other strains.
    • Systems containing the arabinose promoter would not be able to be induced to higher copy in the special strain without also affecting system behavior.
    • This option has the advantage that parts can be prepped from the F plasmid.
    • Could not easily make use of ccdB as a selection marker because DB3.1 does not express the traF gene necessary for expression at high copy. sacB is an alternative but requires sucrose in the media.
  • pUC19 origin in the BioBricks insertion site
    • simply inserting a pUC backbone into the BioBricks insertion site enables the plasmid to be prepped easily and does not introduce any incompatibility issues.
• What is the best way to assemble a pUC backbone with the F plasmid backbone such that the pUC backbone is flanked by BioBricks sites?
  • PCR
  • Use special restriction sites for vector construction (Austin's idea). Expanding on this, we could define a new idempotent assembly standard for exclusive use for vector components.

To be determined

• Are we sure that F plasmids are really at 1-2 copies per cell? Why was pSB2K3-1 measured to be higher than that?
• What parts of the F plasmid are responsible for integration onto the genome? Can we omit them?
• Many of the existing BACs only seem to have a partial sopC CDS, do we want the rest?
  • pSMART VC vector appears to have a more complete sopC region. This may lead to tighter control of copy number.
• A set of orthogonal single copy replication origins to allow multiple vectors to be maintained in a cell. Can we have a set of vectors with F and P1 origins?--BC 17:36, 31 Oct 2005 (EST)
  • Not sure this is possible. I believe the P1 origins use the par set of genes to maintain single copy whereas the F origins use the sop set of genes. The two sets are pretty homologous to eachother and therefore likely incompatible. I need to check on this more. -- RS
  • Perhaps derivatives from the two plasmids the Berkeley iGEM team used might permit two single copy vectors to be used simultaneously. -- RS