Synthetic Biology:Vectors/Single copy plasmid: Difference between revisions

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(updated to reflect current status)
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==Design==
==Design==
The F plasmid origin needs to be designed.  The complete F plasmid with partitioning genes in ~10kb in length.  It contains several BioBricks restriction sites in both coding and noncoding regions.


===Layout===
Once designed, the F plasmid origin can be assembled with an antibiotic resistance marker and cloned into the [[Synthetic Biology:Vectors/pSB**5 design|vector scaffold]] to generate a new single copy BioBricks plasmid.
 
<b>
5' BBa_P1011 (ccdB) -- BBa_I50020 (hc ori)--
 
BBa_G00001 (BB suffix) -- BBa_B0044 (TOPO site) -- BBa_B0042 (translational stop sequence) -- BBa_B0054 (terminator) -- BBa_G00102 (VR) -- BBa_B0062 (rrnC terminator) -- <BBa_P1000 or BBa_P1001 or BBa_P1003 (CmR or TetR or KanR)< -- <BBa_I50000 (F plasmid)< -- BBa_B0053 (His terminator) --
 
BBa_G00100 (VF2) -- BBa_B0055 (terminator) -- BBa_B0042 (translational stop sequence) -- BBa_B0043 (TOPO site) -- BBa_G00000 (BB prefix) 3'
</b>
 
===Ordering information===
 
*See [[Synthetic Biology:Vectors/Parts/Synthesis order]] for ordering information on individual parts.
 
===Proposed features===
*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.
<biblio>
#VanReeth-Biotechniques-1998 pmid=9821593
#Bernard-Gene-1994 pmid=7926841
</biblio>
*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 ''lacZ&alpha;'' 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:Synthetic Biology:Vectors/Single copy plasmid | 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.


==Notes==
===Drawbacks===
===Drawbacks===
*Can only be used in F<sup>-</sup> strains
*Can only be used in F<sup>-</sup> strains
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*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.
*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==
==Relevant pages==
 
===To do list===
*Design unique identifiers for vectors: [[Synthetic Biology:Vectors/Barcode | a plasmid barcode]].  <font color=red> CANCELLED </font>
*Add 7bp site rarely found in ''E. coli'' as part of unique primer binding site.
 
 
 
==Notes==


See the [[Synthetic Biology:Vectors/Parts | list of parts for plasmid engineering]].
See the [[Synthetic Biology:Vectors/Parts | list of parts for plasmid engineering]].

Revision as of 15:38, 17 August 2006

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

The F plasmid origin needs to be designed. The complete F plasmid with partitioning genes in ~10kb in length. It contains several BioBricks restriction sites in both coding and noncoding regions.

Once designed, the F plasmid origin can be assembled with an antibiotic resistance marker and cloned into the vector scaffold to generate a new single copy BioBricks plasmid.

Notes

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.

Relevant pages

See the list of parts for plasmid engineering.

See notes on bacterial artificial chromosomes.

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

Vectors has a lot of general information on vectors.

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