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. Positive selection vectors to generate fused genes for the expression of his-tagged proteins. Biotechniques. 1998 Nov;25(5):898-904.
- Bernard P, Gabant P, Bahassi EM, and Couturier M. Positive-selection vectors using the F plasmid ccdB killer gene. Gene. 1994 Oct 11;148(1):71-4.
- 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.
See notes on bacterial artificial chromosomes.
Vectors has a lot of general information on vectors.