Plasmid backbones/Operation

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Plasmid backbones/Operation
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Reshma's low and medium copy plasmid backbonesAdditional low and medium copy plasmid backbones
Inducible copy number plasmid backbonesMulti-host plasmid backbonesEukaryotic plasmid backbones
BioBrickbackbonevspart.png

Plasmids are circular, double-stranded DNA molecules typically containing a few thousand base pairs that replicate within the cell independently of the chromosomal DNA. Plasmid DNA is easily purified from cells, manipulated using common lab techniques and incorporated into cells. Most BioBrick parts in the Registry are maintained and propagated on plasmids. Thus, construction of BioBrick parts, devices and systems usually requires working with plasmids.

Note: In the Registry, plasmids are made up of two distinct components:

  1. the BioBrick part, device or system that is located in the BioBrick cloning site, between (and excluding) the BioBrick prefix and suffix.
  2. the plasmid backbone which propagates the BioBrick part. The plasmid backbone is defined as the sequence beginning with the BioBrick suffix, including the replication origin and antibiotic resistance marker, and ending with the BioBrick prefix. [Note that the plasmid backbone itself can be composed of BioBrick parts.]

Many BioBrick parts in the Registry are maintained on more than one plasmid backbone!

Once you have assembled your BioBrick device or system, you're then ready to "run" it in your chassis of choice. You might be interested in characterizing its performance, demo'ing your engineered systems to your friends, or documenting its performance for the iGEM jamboree or a publication.

Regardless, it is oftentimes helpful to operate your system on a low or medium copy plasmid backbone. By lowering the DNA copy number of your BioBrick device or system, it will consume less of the cell's resources and thus you can operate more devices in parallel and potentially achieve more reliable system performance.

Note, however, that low or medium copy plasmid backbones tend to have reduced resource consumption because fewer transcripts and proteins are made. Thus, characterization of device or system performance can be more difficult, since you are measuring a smaller number of molecules.

Reshma's low and medium copy operation plasmid backbones

BioBrickoperationvector.png

All of these "operation" plasmid backbones have a common set of features.

  1. A complete BioBrick® cloning site for easy cloning and assembly of BioBrick® parts.
  2. A low or medium copy replication origin to reduce consumption of cellular resources during device or system operation.
  3. Forward and reverse terminators both upstream and downstream of the BioBrick® cloning site to insulate the vector from read-through transcription originating in the cloned BioBrick® device or system and to insulate the cloned BioBrick® system from the vector.
  4. Stop codons in all reading frames to prevent inadvertent translation into or out of the BioBrick cloning site.
  5. Primer binding sites for the standard BioBrick® verification primers VF2 (BBa_G00100) and VR (BBa_G00101). These primers are located for convenient sequencing and screening by colony PCR of cloned BioBrick® devices and systems.

Plasmid backbones are distributed by the Registry with a default insert. There are just a handful of default plasmid inserts used in the Registry. All of the available plasmid backbones include the RFP expression cassette (BBa_J04450) as the default plasmid insert within the BioBrick® cloning site. The RFP cassette enables red/white screening of colonies and ensures that when assembling two BioBrick® parts together, the uncut plasmid (or a plasmid that get the RFP cassette as insert) will be red on the plate whereas the correct colonies will be white.

Plasmid backbones with the plasmid insert of BBa_I52002 also include a high copy replication origin in the default insert. Thus, these plasmid backbones are easily purified in large quantities. Cloning or assembly of a BioBrick® part into the BioBrick® cloning site of the plasmid backbone eliminates the default insert returning the plasmid to the control of the replication origin in the plasmid backbone.


More...
NameDescriptionResistanceRepliconCopy
number
ChassisLength
BBa_K1362095pSB4A50: Low copy BioBrick cloning/expression backbone carrying Amp resistanceApSC101~5 3446
BBa_K1362096pSB4C50: Low(?) copy BioBrick cloning/expression backbone carrying Cm resistanceCpSC101unsure 3408
BBa_K1362097Low copy BioBrick expression backbone carrying Kan resistanceKpSC101~5 3470
BBa_K864000Low copy BioBrick standard vectorApSC101~5 3639
BBa_K864001Low copy BioBrick standard vectorCpSC101~5 3466
BBa_K864002Low copy BioBrick standard vectorKpSC101~5 3663
BBa_K864003Low copy BioBrick standard vectorSpSC101~5 3793
BBa_K864004Low copy BioBrick standard vectorApSC101~5 3708
BBa_K864005Low copy BioBrick standard vectorCpSC101~5 3534
BBa_K864006Low copy BioBrick standard vectorKpSC101~5 3732
BBa_K864007Low copy BioBrick standard vectorSpSC101~5 3861
BBa_K864008Low copy BioBrick standard vector with LacIApSC101~5 4819
BBa_K864009Low copy BioBrick standard vector with LacICpSC101~5 4646
BBa_K864010Low copy BioBrick standard vector with LacIKpSC101~5 4843
BBa_K864011Low copy BioBrick standard vector with LacISpSC101~5 4973
BBa_K864012Low copy BioBrick standard vector with LacIApSC101~5 4888
BBa_K864013Low copy BioBrick standard vector with LacICpSC101~5 4714
BBa_K864014Low copy BioBrick standard vector with LacIKpSC101~5 4912
BBa_K864015Low copy BioBrick standard vector with LacISpSC101~5 5041
BBa_K864016Low copy BioBrick temperature sensitive standard vectorApSC101ts~5 3639
BBa_K864017Low copy BioBrick temperature sensitive standard vectorCpSC101ts~5 3466
BBa_K864018Low copy BioBrick temperature sensitive standard vectorKpSC101ts~5 3663
BBa_K864019Low copy BioBrick temperature sensitive standard vectorSpSC101ts~5 3793
BBa_K864020Low copy BioBrick temperature sensitive standard vectorApSC101ts~5 3708
BBa_K864021Low copy BioBrick temperature sensitive standard vectorCpSC101ts~5 3534
BBa_K864022Low copy BioBrick temperature sensitive standard vectorKpSC101ts~5 3732
BBa_K864023Low copy BioBrick temperature sensitive standard vectorSpSC101ts~5 3861
pSB3C5Low to medium copy BioBrick standard vectorCp15A10-12 2738
pSB3K5Low to medium copy BioBrick standard vectorKp15A10-12 2936
pSB3T5Low to medium copy BioBrick standard vectorTp15A10-12 3252
pSB4A5Low copy BioBrick standard vectorApSC101~5 3395
pSB4C5Low copy BioBrick standard vectorCpSC101~5 3221
pSB4K5Low copy BioBrick standard vectorKpSC101~5 3419
pSB4T5Low copy BioBrick standard vectorTpSC101~5 3735


ReshmaShettyPhoto.jpg
DrewEndyPhoto.jpeg
TomKnightPhoto.jpg
Reshma Shetty, Drew Endy, and Tom Knight constructed this set of plasmid backbones. You can read more about the design and construction of these plasmid backbones in their open-access paper, Engineering BioBrick vectors from BioBrick parts published in the Journal of Biological Engineering.


Additional low and medium copy operation plasmid backbones

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There are additional low and medium copy BioBrick® plasmid backbones available, all of which have a complete BioBrick cloning site.

StefanLuziPhoto.jpg
Stefan Luzi constructed the three plasmid backbones (pSB6A1, pSB4C1, and pSB3K1) as a member of the 2007 ETH Zurich iGEM team.
JeffTaborPhoto.jpg
Jeff Tabor constructed plasmid backbone BBa_J64100 in Chris Voigt's lab.
ReshmaShettyPhoto.jpg
Reshma Shetty constructed plasmid backbone pSB3K3 in Tom Knight's lab.


More...
NameDescriptionResistanceRepliconCopy
number
ChassisLength
BBa_J61031Amplifiable BAC plasmidKColE1/R6K/F  10662
BBa_K125000Broad host range BioBrick plasmid derived from pRL1383a with low to medium copy number    9047
BBa_K592200Low to medium copy BioBrick standard vectorSp15A10-12 3137
pSB3K1pACYC177 converted into BioBrick vector (formerly BBa_I739202)Kp15A10-12 3064
pSB3K3Low to medium copy BioBrick standard vectorKp15A10-12 2750
pSB4C1pCK01 converted into BioBrick vector (formerly BBa_I739202)CpSC1015-12 3155
pSB6A1pBR322 converted to BioBrick vector (formally BBa_I739201)ApMB115-20 4022


Inducible copy number operation plasmid backbones

BioBrickinduciblecopyoperationvector.png

Sometimes it is nice to have the best of both worlds. For example, you'd usually like a BioBrick® part to propagate at low copy, but you'd also like to switch the part over to high copy when you're interested in purifying plasmid DNA. The solution is an inducible copy number plasmid backbone. Both plasmid backbones listed below have a high copy replication origin under the control of a LacI-repressible promoter. When the plasmid is propagated in an E. coli strain that expresses the repressor lacI in high quantities, such as D1210 (or BBa_V1003), the plasmid is maintained at low copy by the F' origin. To increase the plasmid copy number, simply add the small molecule IPTG to induce the LacI-repressible promoter controlling the high copy number origin.

  • Note: because these inducible copy number vectors rely on LacI for control of the high copy number origin, they are incompatible with any BioBrick® part, device, or system that include lacI or a LacI-regulatable promoter.

The plasmid backbones listed below have a common set of features.

  1. A complete BioBrick® cloning site for easy cloning and assembly of BioBrick parts.
  2. Terminators flanking the BioBrick® cloning site to insulate the vector from read-through transcription originating in the cloned BioBrick® part, device or system.
  3. Primer binding sites for the standard BioBrick® verification primers VF2 (BBa_G00100) and VR (BBa_G00101). These primers are located for convenient sequencing and screening by colony PCR of cloned BioBrick® parts, devices, and systems.

Plasmid backbones are distributed by the Registry with a default insert. There are just a handful of default plasmid inserts used in the Registry. Many the available plasmid backbones have the ccdB positive selection marker (BBa_P1010) as the default plasmid insert within the BioBrick® cloning site. The ccdB gene ensures that when assembling two BioBrick® parts together, the uncut plasmid is not transformed. However, inclusion of the ccdB gene means that these vectors must be propagated in a ccdB tolerant strain, such as E. coli strain DB3.1 (BBa_V1005).


More...
NameDescriptionResistanceRepliconCopy
number
ChassisLength
pSB2K3Inducible copy number BioBrick plasmidKF' and P1 lytic<10 or >100lacIq like BBa_V10034425
pSB2K4pSB2K3-derived BioBrick plasmid free of many restriction sitesKF' and P1 lytic<10 or >100lacIq like BBa_V10034467


SriKosuriPhoto.jpg
LeonChanPhoto.jpg
AustinChePhoto.jpg
Sri Kosuri and Leon Chan constructed plasmid backbone pSB2K3 in Drew Endy's lab based on the pSCANS vector from Brookhaven National Lab. They, together with Austin Che, constructed pSB2K4.

Multi-host operation plasmid backbones

SarahHollingsheadPhoto.jpg
Sarah Hollingshead constructed this multi-host BioBrick plasmid backbone as a member of the 2007 University of Edinburgh iGEM team from the pTG262 vector generously provided by Dr Mike Gasson & Dr. Claire Shearman of the Institute of Food Research in Norwich. The Edinburgh team used the plasmid backbone as a shuttle plasmid backbone between lactic acid bacteria and E. coli. It has a gram positive replication origin.


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Eukaryotic operation plasmid backbones

MonikaCiglicPhoto.jpg
Monika Ciglic constructed this E. coli high copy plasmid backbone with a eukaryotic terminator as a member of the 2006 Slovenija iGEM team from pSB1AK3.


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Notes

References

<biblio>

  1. Hershfield74 pmid=4610576
  2. Mandecki90 pmid=2227445
  3. Viera82 pmid=6295879
  4. YanishPerron85 pmid=2985470
  5. Messing91 pmid=2055478
  6. Chang74 pmid=4598290
  7. Cohen77 pmid=334752
  8. Cabello76 pmid=765836
  9. Sugiura93 pmid=8376344
  10. Miller95 pmid=7665462
  11. Bolivar77 pmid=344136
  12. Bolibar77b pmid=344137
  13. Chang78 pmid=149110
  14. Murakami87 pmid=3031019
  15. Uga89 pmid=10393200
  16. Kawasaki96 pmid=9003285
  17. Stueber82 pmid=6327267
  18. Bernard84 pmid=7926841

</biblio>