Synthetic Biology:Vectors/Single copy plasmid

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==Design==
==Design==
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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.
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===Layout===
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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.
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<b>
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Chris Anderson suggested inclusion of the R6K origin in these plasmids (rather than inclusion of a pUC19 origin in the multiple cloning site).  The R6K origin is a conditional origin.  It only works in the presence of the trans-acting protein &Pi; (encoded by pir) for replication.  R6K replicates at a medium copy (15 per cell) in pir+ strains and high copy (250 per cell) in pir-116 (high-copy-number mutant) E. coli hosts.
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5' BBa_P1010 (ccd) -- BBa_I50020 (hc ori)--  
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BBsuffix -- BBa_B0044 (TOPO site) -- BBa_B0042 (translational stop sequence) -- [[Synthetic Biology:Vectors/Barcode | plasmid barcode]] -- BBa_B0054 (terminator) -- BBa_G00102 (VR) -- 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_I50000 (F plasmid, '''switch orientation''')
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==Fabrication==
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BBa_G00100 (VF2) -- BBa_B0055 (terminator) -- [[Synthetic Biology:Vectors/Barcode | plasmid barcode]] -- BBa_B0042 (translational stop sequence) -- BBa_B0043 (TOPO site) -- BB prefix 3'
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*'''[[User:Rshetty|Reshma]] 15:27, 27 February 2007 (EST)''': Tom suggested that mutations of the BioBricks sites could be done via Pete Carr, Farren Isaacs and George Church's single stranded mutation method.
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</b>
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'''Total length''' (excluding scars and barcode, including prefix and suffix)
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*CmR version:  8296bp
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*TetR version: 8786bp
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*KanR version: 8500bp
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'''Total length''' (excluding barcode, including prefix, suffix and barcodes)
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*CmR version:  8392bp
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*TetR version: 8882bp
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*KanR version: 8596bp
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===Ordering information===
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*See [[Synthetic Biology:Vectors/Parts]] for ordering information on individual parts.
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===Proposed features===
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*F plasmid backbone
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*positive selection marker (i.e. ''ccdB'' or ''sacB'') in between BioBricks restrictions sites to facilitate cloning
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*a high copy origin in the multiple cloning site to enable easy purification of the vector
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*strong terminators flanking the BioBricks insertion site
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*no loxP or cos insertion sites or Tn7 attachment sites? 
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**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?
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*no blue-white screening? 
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**inclusion of a ''lacZ&alpha;'' fragment would restrict its use as a part
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*multiple versions with different antibiotic resistance markers
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*no selection system for mammalian cells
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*VF2 and VR sites
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*unique tag near but outside the cloning sites for identification during sequencing. (from Randy)
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*resistance markers
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**orient the ampicillin antibiotic resistance cassette on the reverse strand from the BioBricks insertion site
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**every plasmid should have AmpR plus another resistance marker.
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**need to include a terminator downstream of the antibiotic resistance cassette (use a terminator from the original BioBricks plasmids)
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*[[Topoisomerase I mediated TA cloning]]
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*include a sequence with translational stops in all frames flanking each side of the MCS
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*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.
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*the plasmid origin transcripts should be oriented in the forward direction such that readthrough from the origin can't affect the insert.
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*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.
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==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.
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==Planning==
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==Relevant pages==
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===Current status===
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The following parts have been designed
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*<bbpart>BBa_I50000</bbpart>: F plasmid backbone with BioBricks restriction sites removed
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*<bbpart>BBa_I50020</bbpart>: high copy origin from pSB1A3
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*<bbpart>BBa_B0055</bbpart>: upstream flanking terminator
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*<bbpart>BBa_B0054</bbpart>: downstream flanking terminator
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*<bbpart>BBa_B0053</bbpart>: bidirectional terminator from ''E. coli'' his operon
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*<bbpart>BBa_B0062</bbpart>: reverse terminator
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*<bbpart>BBa_P1002</bbpart>: cassette providing ampicillin resistance (from [[Tom Knight]])
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*<bbpart>BBa_P1003</bbpart>: cassette providing kanamycin resistance (from [[Tom Knight]])
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*<bbpart>BBa_P1004</bbpart>: cassette providing ampicillin resistance in reverse orientation.
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*<bbpart>BBa_P1005</bbpart>: cassette providing tetracycline resistance and ampicillin resistance with terminators.
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*<bbpart>BBa_P1006</bbpart>: cassette providing chloramphenicol resistance and ampicillin resistance with terminators.
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*<bbpart>BBa_P1007</bbpart>: cassette providing kanamycin resistance and ampicillin resistance with terminators.
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*<bbpart>BBa_B0042</bbpart>: translational stop sequence. (see [[Non-functional DNA sequences]])
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*<bbpart>BBa_B0043</bbpart>: forward Topoisomerase I cloning site. (see [[Topoisomerase I mediated TA cloning]])
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*<bbpart>BBa_B0043</bbpart>: reverse Topoisomerase I cloning site. (see [[Topoisomerase I mediated TA cloning]])
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The following parts have been designed, fabricated and tested
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*<bbpart>BBa_P1001</bbpart>: cassette providing tetracycline resistance (from [[Austin Che]])
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*<bbpart>BBa_P1000</bbpart>: cassette providing chloramphenicol resistance (from [[Austin Che]])
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*<bbpart>BBa_I1000</bbpart> or <bbpart>BBa_P1010</bbpart>: ccd operon in BioBricks format (from Leon Chan)
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===To do list===
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*Design unique identifiers for vectors: [[Synthetic Biology:Vectors/Barcode | a plasmid barcode]].
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*Specify assembled plasmid in registry.
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**Specify multiple cloning site modules?
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*Add 7bp site rarely found in ''E. coli'' as part of unique primer binding site.
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*Draw up the parts with size information.
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*Redesign parts such that replication origins and antibiotic resistance markers can be swapped out with unique restriction enzymes.
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*If certain parts are being synthesized, then they should be stripped of enzyme recognition sites (i.e. replication origins and antibiotic resistance markers).
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===To be decided===
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*Choose between manual assembly of vector modules or direct synthesis of all plasmid variants
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**Can we get a price break for synthesizing multiple plasmid variants?
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**How many assemblies would we need to do?
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**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?--[[User:Bcanton|BC]]
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***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. --[[Reshma Shetty | RS]]
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*If all the vector components are specified in BioBricks format, how do we construct a BioBricks insertion site?
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**Blunt-end ligation?
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**Other restriction enzyme sites?
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**PCR
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**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.
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*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.
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*Should we allow swapping of just one of the resistance markers (i.e. cmR/tetR/kanR) or both (i.e. ampR + another)?
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*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.
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===To be determined===
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*Are we sure that F plasmids are really at 1-2 copies per cell?  Why was pSB2K3-1 measured to be higher than that?
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**From Johann Paulsson: it is unclear how tight of control F plasmid based vectors have over copy number fluctuation.  Having the vector exist at single copy strongly depends on generation time.  Faster growing cells are more likely to have multiple overlapping rounds of replications occurring simultaneously.
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*What parts of the F plasmid are responsible for integration onto the genome?  Can we omit them?
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**cos and/or loxP sites are generally used for integration in the genome.  Currently, I have no plans to include them in this vector.
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*Many of the existing BACs only seem to have a partial ''sopC'' CDS, do we want the rest?
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**pSMART VC vector appears to have a more complete ''sopC'' region.  This may lead to tighter control of copy number.
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*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?--[[User:Bcanton|BC]] 17:36, 31 Oct 2005 (EST)
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**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.  --[[Reshma Shetty | RS]]
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** Perhaps derivatives from the two plasmids the Berkeley iGEM team used might permit two single copy vectors to be used simultaneously. --[[Reshma Shetty | RS]]
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* Should the flanking terminators be placed outside the VF2 and VR primer binding sites?  Is it useful to have them within?  Moving the flanking terminators outside the primer binding sites means fewer bases to sequence through before hitting the part.  Alternatively, we could move the terminators to just inside the primers since anyway ~40bp are needed before sequence data is of high quality.
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**Tom thinks this doesn't matter but suggests including some translational stops around the multiple cloning site
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==Notes==
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See the [[Synthetic Biology:Vectors/Parts | list of parts for plasmid engineering]].
See the [[Synthetic Biology:Vectors/Parts | list of parts for plasmid engineering]].
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[[Category:Project]]
[[Category:Project]]
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==References==
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<biblio>
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#Jones-BiotechnolBioeng-1998 pmid=10099385
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#Metcalf-Gene-1994 pmid=8125283
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</biblio>

Current revision

Contents

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.

Chris Anderson suggested inclusion of the R6K origin in these plasmids (rather than inclusion of a pUC19 origin in the multiple cloning site). The R6K origin is a conditional origin. It only works in the presence of the trans-acting protein Π (encoded by pir) for replication. R6K replicates at a medium copy (15 per cell) in pir+ strains and high copy (250 per cell) in pir-116 (high-copy-number mutant) E. coli hosts.

Fabrication

  • Reshma 15:27, 27 February 2007 (EST): Tom suggested that mutations of the BioBricks sites could be done via Pete Carr, Farren Isaacs and George Church's single stranded mutation method.

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.

References

  1. Jones KL and Keasling JD. . pmid:10099385. PubMed HubMed [Jones-BiotechnolBioeng-1998]
  2. Metcalf WW, Jiang W, and Wanner BL. . pmid:8125283. PubMed HubMed [Metcalf-Gene-1994]
All Medline abstracts: PubMed HubMed
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