Synthetic Biology:Vectors/Single copy plasmid

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==Design==
==Design==
 +
 +
===Layout===
<b>
<b>
-
5' BBa_P1010 (ccd) -- BBa_I50020 --  
+
5' BBa_P1010 (ccd) -- BBa_I50020 (hc ori)--  
BBsuffix -- BBa_B0044 (TOPO site) -- BBa_B0042 (translational stop sequence) -- [[Synthetic Biology:Vectors/Barcode | plasmid barcode]] -- BBa_B0054 (terminator) -- BBa_G00102 (VR) -- BBa_I50000 (F plasmid) -- BBa_P1000 or BBa_P1001 or BBa_P1003 (TetR or CmR or KanR) -- BBa_B0053 (His terminator) -- BBa_B0062 (reverse rrnC terminator) -- BBa_P1004 (reverse AmpR) --  
BBsuffix -- BBa_B0044 (TOPO site) -- BBa_B0042 (translational stop sequence) -- [[Synthetic Biology:Vectors/Barcode | plasmid barcode]] -- BBa_B0054 (terminator) -- BBa_G00102 (VR) -- BBa_I50000 (F plasmid) -- BBa_P1000 or BBa_P1001 or BBa_P1003 (TetR or CmR or KanR) -- BBa_B0053 (His terminator) -- BBa_B0062 (reverse rrnC terminator) -- BBa_P1004 (reverse AmpR) --  
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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'
BBa_G00100 (VF2) -- BBa_B0055 (terminator) -- [[Synthetic Biology:Vectors/Barcode | plasmid barcode]] -- BBa_B0042 (translational stop sequence) -- BBa_B0043 (TOPO site) -- BB prefix 3'
</b>
</b>
 +
 +
===Ordering information===
 +
 +
{| border="1"
 +
|-
 +
| '''Part number'''
 +
| '''Description'''
 +
| '''Size'''
 +
| '''BioBrick available'''
 +
| '''Template available'''
 +
| '''Want synthesized'''
 +
|-
 +
| colspan="6" | '''BASIC PARTS'''
 +
|--
 +
| BBa_P1010
 +
| ccd
 +
| 675bp
 +
| Yes
 +
| Yes
 +
| No
 +
|--
 +
| BBa_I50020
 +
| high copy origin
 +
| 858bp
 +
| No
 +
| Yes
 +
| Maybe
 +
|--
 +
| colspan="6" |
 +
|--
 +
|
 +
| BBsuffix
 +
| 21bp
 +
| No
 +
| No
 +
| No (can make with primers)
 +
|--
 +
| BBa_B0044
 +
| TOPO site
 +
| 13bp
 +
| No
 +
| No
 +
| No (can make with primers)
 +
|--
 +
| BBa_B0042
 +
| translational stop sequence
 +
| 12bp
 +
| No
 +
| No
 +
| No (can make with primers)
 +
|--
 +
|
 +
| [[Synthetic Biology:Vectors/Barcode | plasmid barcode]]
 +
|
 +
|
 +
|
 +
|
 +
|--
 +
| BBa_B0054
 +
| terminator
 +
| 69bp
 +
| No
 +
| No
 +
| ? (maybe make with primer extension)
 +
|--
 +
| BBa_G00102
 +
| VR
 +
| 20bp
 +
| No
 +
| No
 +
| No (can make with primers)
 +
|--
 +
| BBa_I50000
 +
| F plasmid
 +
| 4640bp
 +
| No
 +
| No
 +
| Yes (has multiple BioBricks sites)
 +
|--
 +
| BBa_P1000
 +
| CmR
 +
| 789bp
 +
| Yes (Austin)
 +
| Yes
 +
| No
 +
|--
 +
| BBa_P1001
 +
| TetR
 +
| 1279bp
 +
| Yes (Austin)
 +
| Yes
 +
| No
 +
|--
 +
| BBa_P1003
 +
| KanR
 +
| 993bp
 +
| Maybe (TK)
 +
| Yes
 +
| Maybe
 +
|--
 +
| BBa_B0053
 +
| His terminator
 +
| 72bp
 +
| No
 +
| No
 +
| Yes (previous synthesis attempt via primer extension failed)
 +
|--
 +
| BBa_B0062
 +
| reverse rrnC terminator
 +
| 41bp
 +
| No
 +
| No
 +
| Maybe (maybe make with primer extension, but terminators can be hard)
 +
|--
 +
| BBa_P1004
 +
| reverse AmpR
 +
| 941bp
 +
| No
 +
| Yes
 +
| Maybe
 +
|--
 +
| BBa_G00100
 +
| VF2
 +
| 20bp
 +
| No
 +
| No
 +
| No (can make with primers)
 +
|--
 +
| BBa_B0055
 +
| terminator
 +
| 78bp
 +
| No
 +
| No
 +
| Maybe (maybe make with primer extension, but terminators can be hard)
 +
|--
 +
|
 +
| [[Synthetic Biology:Vectors/Barcode | plasmid barcode]]
 +
|
 +
|
 +
|
 +
|
 +
|--
 +
| BBa_B0042
 +
| translational stop sequence
 +
| 12bp
 +
| No
 +
| No
 +
| No (can make with primers)
 +
|--
 +
| BBa_B0043
 +
| forward TOPO site
 +
| 13p
 +
| No
 +
| No
 +
| No (can make with primers)
 +
|--
 +
|
 +
| BB prefix
 +
| 22bp
 +
| No
 +
| No
 +
| No (can make with primers)
 +
|--
 +
| colspan="6" | '''COMPOSITE PARTS'''
 +
|--
 +
| BBa_P1005
 +
| tetR and ampR
 +
| 1867bp
 +
| No
 +
| No
 +
| Maybe
 +
|--
 +
| BBa_P1006
 +
| cmR and ampR
 +
| 2357bp
 +
| No
 +
| No
 +
| Maybe
 +
|--
 +
| BBa_P1007
 +
| CmR and ampR
 +
| 2071bp
 +
| No
 +
| No
 +
| Maybe
 +
|}
===Proposed features===
===Proposed features===

Revision as of 19:30, 9 March 2006

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

Layout

5' BBa_P1010 (ccd) -- BBa_I50020 (hc ori)--

BBsuffix -- BBa_B0044 (TOPO site) -- BBa_B0042 (translational stop sequence) -- plasmid barcode -- BBa_B0054 (terminator) -- BBa_G00102 (VR) -- BBa_I50000 (F plasmid) -- BBa_P1000 or BBa_P1001 or BBa_P1003 (TetR or CmR or KanR) -- BBa_B0053 (His terminator) -- BBa_B0062 (reverse rrnC terminator) -- BBa_P1004 (reverse AmpR) --

BBa_G00100 (VF2) -- BBa_B0055 (terminator) -- plasmid barcode -- BBa_B0042 (translational stop sequence) -- BBa_B0043 (TOPO site) -- BB prefix 3'

Ordering information

Part number Description Size BioBrick available Template available Want synthesized
BASIC PARTS
BBa_P1010 ccd 675bp Yes Yes No
BBa_I50020 high copy origin 858bp No Yes Maybe
BBsuffix 21bp No No No (can make with primers)
BBa_B0044 TOPO site 13bp No No No (can make with primers)
BBa_B0042 translational stop sequence 12bp No No No (can make with primers)
plasmid barcode
BBa_B0054 terminator 69bp No No  ? (maybe make with primer extension)
BBa_G00102 VR 20bp No No No (can make with primers)
BBa_I50000 F plasmid 4640bp No No Yes (has multiple BioBricks sites)
BBa_P1000 CmR 789bp Yes (Austin) Yes No
BBa_P1001 TetR 1279bp Yes (Austin) Yes No
BBa_P1003 KanR 993bp Maybe (TK) Yes Maybe
BBa_B0053 His terminator 72bp No No Yes (previous synthesis attempt via primer extension failed)
BBa_B0062 reverse rrnC terminator 41bp No No Maybe (maybe make with primer extension, but terminators can be hard)
BBa_P1004 reverse AmpR 941bp No Yes Maybe
BBa_G00100 VF2 20bp No No No (can make with primers)
BBa_B0055 terminator 78bp No No Maybe (maybe make with primer extension, but terminators can be hard)
plasmid barcode
BBa_B0042 translational stop sequence 12bp No No No (can make with primers)
BBa_B0043 forward TOPO site 13p No No No (can make with primers)
BB prefix 22bp No No No (can make with primers)
COMPOSITE PARTS
BBa_P1005 tetR and ampR 1867bp No No Maybe
BBa_P1006 cmR and ampR 2357bp No No Maybe
BBa_P1007 CmR and ampR 2071bp No No Maybe

Proposed features

  • F plasmid backbone
  • positive selection marker (i.e. ccdB or sacB) in between BioBricks restrictions sites to facilitate cloning
  • 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.

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

  • <bbpart>BBa_I50000</bbpart>: F plasmid backbone with BioBricks restriction sites removed
  • <bbpart>BBa_I50020</bbpart>: high copy origin from pSB1A3
  • <bbpart>BBa_B0055</bbpart>: upstream flanking terminator
  • <bbpart>BBa_B0054</bbpart>: downstream flanking terminator
  • <bbpart>BBa_B0053</bbpart>: bidirectional terminator from E. coli his operon
  • <bbpart>BBa_B0062</bbpart>: reverse terminator
  • <bbpart>BBa_P1002</bbpart>: cassette providing ampicillin resistance (from Tom Knight)
  • <bbpart>BBa_P1003</bbpart>: cassette providing kanamycin resistance (from Tom Knight)
  • <bbpart>BBa_P1004</bbpart>: cassette providing ampicillin resistance in reverse orientation.
  • <bbpart>BBa_P1005</bbpart>: cassette providing tetracycline resistance and ampicillin resistance with terminators.
  • <bbpart>BBa_P1006</bbpart>: cassette providing chloramphenicol resistance and ampicillin resistance with terminators.
  • <bbpart>BBa_P1007</bbpart>: cassette providing kanamycin resistance and ampicillin resistance with terminators.
  • <bbpart>BBa_B0042</bbpart>: translational stop sequence. (see Non-functional DNA sequences)
  • <bbpart>BBa_B0043</bbpart>: forward Topoisomerase I cloning site. (see Topoisomerase I mediated TA cloning)
  • <bbpart>BBa_B0043</bbpart>: reverse Topoisomerase I cloning site. (see Topoisomerase I mediated TA cloning)

The following parts have been designed, fabricated and tested

  • <bbpart>BBa_P1001</bbpart>: cassette providing tetracycline resistance (from Austin Che)
  • <bbpart>BBa_P1000</bbpart>: cassette providing chloramphenicol resistance (from Austin Che)
  • <bbpart>BBa_I1000</bbpart> or <bbpart>BBa_P1010</bbpart>: ccd operon in BioBricks format (from Leon Chan)

To do list

  • Design unique identifiers for vectors: a plasmid barcode.
  • Specify assembled plasmid in registry.
    • Specify multiple cloning site modules?
  • Add 7bp site rarely found in E. coli as part of unique primer binding site.
  • Draw up the parts with size information.
  • Redesign parts such that replication origins and antibiotic resistance markers can be swapped out with unique restriction enzymes.

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 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?
    • 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.
  • 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
  • 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.
    • Tom thinks this doesn't matter but suggests including some translational stops around the multiple cloning site

Notes

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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