Synthetic Biology:Vectors/Single copy plasmid
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
- Copy number is uncertain or variable making it difficult to infer PoPS per DNA copy.
- 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.
5' BBa_P1011 (ccdB) -- BBa_I50020 (hc ori)--
BBa_G00001 (BB suffix) -- BBa_B0044 (TOPO site) -- BBa_B0042 (translational stop sequence) -- plasmid barcode -- BBa_B0054 (terminator) -- BBa_G00102 (VR) -- BBa_B0045 (NheI) -- BBa_P1000 or BBa_P1001 or BBa_P1003 (CmR or TetR or KanR) -- BBa_B0053 (His terminator) -- BBa_P1002 (AmpR) -- BBa_B0062 (rrnC terminator) -- BBa_B0046 (NsiI) -- BBa_B0047 (MfeI) -- BBa_I50001 (F plasmid, reverse) -- BBa_B0048 (AvrII) --
BBa_G00100 (VF2) -- BBa_B0055 (terminator) -- plasmid barcode -- BBa_B0042 (translational stop sequence) -- BBa_B0043 (TOPO site) -- BBa_G00000 (BB prefix) 3'
Total length (excluding scars and barcode, including prefix and suffix)
- CmR version: 8296bp
- TetR version: 8786bp
- KanR version: 8500bp
Total length (excluding barcode, including prefix, suffix and barcodes)
- CmR version: 8392bp
- TetR version: 8882bp
- KanR version: 8596bp
- See Synthetic Biology:Vectors/Parts for ordering information on individual parts.
- F plasmid backbone
- negative selection marker (i.e. ccdB or sacB) in between BioBricks restrictions sites to facilitate cloning
- Actually, ccdB is often called a positive selection marker.
- Van Reeth T, Drèze PL, Szpirer J, Szpirer C, and Gabant P. . pmid:9821593.
- Bernard P, Gabant P, Bahassi EM, and Couturier M. . pmid:7926841.
- a high copy origin in the multiple cloning site to enable easy purification of the vector
- 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)
- resistance markers
- orient the ampicillin antibiotic resistance cassette on the reverse strand from the BioBricks insertion site
- every plasmid should have AmpR plus another resistance marker.
- need to include a terminator downstream of the antibiotic resistance cassette (use a terminator from the original BioBricks plasmids)
- Topoisomerase I mediated TA cloning
- include a sequence with translational stops in all frames flanking each side of the MCS
- apparently when you sequence, the first 30 bp or so are really bad (see also the talk page) but there can also be a bad spot at around base pair 80. So the verification primers should be about 100bp away from the multiple cloning site.
- the plasmid origin transcripts should be oriented in the forward direction such that readthrough from the origin can't affect the insert.
- Enable swapping in and out of different origins and antibiotic resistance cassettes. Note that such a scheme would likely make it difficult to enable/enforce plasmid barcodes. See Synthetic Biology:Vectors/Modular construction scheme for details.
- 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
- Design unique identifiers for vectors: a plasmid barcode. CANCELLED
- Add 7bp site rarely found in E. coli as part of unique primer binding site.
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?
- 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.
- Should we reduce the spacing between the verification primer binding sites and the BioBricks site to 30bp? This may require human intervention to read sequence at around the 60 bp mark. Such a change requires that the flanking terminators be moved outside the verification primer binding sites.
- Should we allow swapping of just one of the resistance markers (i.e. cmR/tetR/kanR) or both (i.e. ampR + another)?
- One risk of permitting the two antibiotic resistance markers to transcribe in opposite directions is that antisense RNA is created to the other resistance marker, rendering it useless. Thus, the resistance markers should perhaps point in the same direction away from the multiple cloning site. Therefore, both resistance markers should likely be on the opposite strand (which would mean they would need to be remade via PCR). Or instead, the origins could be put on the opposite strand.
See notes on bacterial artificial chromosomes.
Vectors has a lot of general information on vectors.