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

Goal

Design and fabricate a single copy vector in which BioBricks devices can be characterized. To date most characterization work has been done in low or high copy vectors which have several issues including

1. Copy number is uncertain or variable making it difficult to infer PoPS per DNA copy.
2. At high copy, devices place a high metabolic load on the cell thereby altering host physiology and observed device behavior.

The proposed solution to these two problems is to characterize devices at single copy in the cell. Obviously, such a vector will vary between 1 and 2 copies per cell over the cell cycle but nevertheless will hopefully present an improvement over the current situation. The advantage of using a single copy plasmid rather than simply integrating the device into the genome is that a separate plasmid offers some isolation from the host and makes moving the device between different host strains slightly easier.

Design

5' -- VF2 -- plasmid barcode -- BBa_B0055 -- BB prefix -- BBa_I1000 -- ?pUC19 backbone? -- BBsuffix -- BBa_B0054 -- VR -- BBa_I50000 -- antibiotic resistance cassette -- 3'

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
• Unique tag near but outside the cloning sites for identification during sequencing. (from Randy)
• orient the antibiotic resistance cassette on the reverse strand from the BioBricks insertion site

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.

Planning

Current status

The following parts have been designed

• BBa_I1000: ccd operon in BioBricks format
• BBa_I50000: F plasmid backbone with BioBricks sites removed
• BBa_I50010: oriV origin which requires TrfA protein to be functional.
• BBa_B0055: upstream flanking terminator
• BBa_B0054: downstream flanking terminator

To do list

• One of the things needed 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.
  • I have TetR and CmR BioBricked using Tom's primers. --Austin 18:26, 3 Dec 2005 (EST)
• Design unique identifiers for vectors.
• Design a pUC19 backbone for possible use in the MCS so that the plasmid can be purified at high copy

To be decided

• Choose between manual assembly of vector modules or direct synthesis of all plasmid variants
  • Can we get a price break for synthesizing multiple plasmid variants?
  • How many assemblies would we need to do?
  • Is there a hybrid approach? Could we PCR the F plasmid backbone since its long and then have the collection of smaller parts (that would otherwise involve several assemblies) synthesized? Maybe a partial synthesis approach would help get around the issue of constructing a BioBricks insertion site?--BC
    • PCR'ing the F plasmid backbone is not very practical since there are several BioBricks sites in the backbone each of which would need to be individually mutated out. It is unlikely that there is anyone who is willing to do this much work. Therefore, the current plan is to synthesize the backbone. -- RS
• 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 a secondary, inducible copy number origin or insert the pUC backbone inside of the BioBricks cloning site to facilitate prepping of the vector?
  • 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.
      • I must not be understanding this, isn't DB3.1 resistant to "ccdB" and hence you wouldn't be using DB3.1 for selection anyway?
        • I was referring to the fact that prepping the plasmid for use would be difficult. I envision using this plasmid not to assemble BioBrick parts but as the vector in which characterization takes place. Therefore, I assume we would more often want to prep the vector (which contains ccdB) than a vector with a part in it. Thus, in order to easily prep large amounts of vector, we would need a host strain that can tolerate ccdB and has the traF copy up mutation which likely does not exist (and therefore would need to be constructed). I am not convinced that we will want to prep parts in the F plasmid so frequently that it is worth the effort of constructing such a strain. -- RS
  • 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.
    • allows the plasmid to be prepped at very high copy
    • parts in the F plasmid cannot be easily prepped
      • So this means we'd have to TempliPhi in order to sequence? The TempliPhi website seems to think this is reliable enough to work--BC
      • Sequencing could be done by either sequencing a PCR product or via Templiphi. You can prep F plasmid from cells, it is just not quite as simple and quick as a miniprep since you get much lower yields. -- RS
• 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?
  • cos and/or loxP sites are generally used for integration in the genome. Currently, I have no plans to include them in this vector.
• 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

Notes

See notes on bacterial artificial chromosomes.

BioBrick plasmid parts

Replication origins

• BBa_I50000: F plasmid backbone with BioBricks sites removed
• BBa_I50010: oriV origin which requires TrfA protein to be functional.
• pUC19 high copy backbone

Antibiotic resistance cassettes

• BBa_P1001: cassette providing tetracycline resistance.
• BBa_P1000: cassette providing chloramphenicol resistance.
• antibiotic resistance cassettes (from Tom)

Terminators

• BBa_B0055: upstream flanking terminator
• BBa_B0054: downstream flanking terminator

Notes

It wasn't clear from the website if these were bi-directional? Not sure that this is very important--BC

I don't know if the terminators are bidirectional. These terminators are used as flanking terminators in other vectors and are claimed to make the cloning of difficult pieces of DNA (like strong promoters) easier. This is why I was planning on orienting the antibiotic resistance cassette in the opposite direction so that read through from upstream of the multiple cloning site is less of an issue. -- RS

Others

• BBa_I1000: ccd operon in BioBricks format
• unique identifier for the vector