Help:Plasmid backbones/Construction

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ReshmaShettyPhoto.jpg DrewEndyPhoto.jpeg TomKnightPhoto.jpg Reshma Shetty, Drew Endy and Tom Knight developed a method and set of parts (available via the Registry) to make the process of building a new BioBrick® plasmid backbone easier. The method and part collection are described in their open-access paper, Engineering BioBrick vectors from BioBrick parts published in the Journal of Biological Engineering.

How to construct a new BioBrick® plasmid backbone

Constructing any new BioBrick® standard construction plasmid starting from the BioBrick® base vector requires just two assembly steps.

Figure 1: Schematic diagram of procedure to construct new BioBrick® vectors from the BioBrick® base vector. Click the image to view a larger schematic diagram describing how to build new BioBrick® vectors from the BioBrick® base vector.
Figure 2: BioBrick® vector parts.
  1. Design your replication origin(s) and antibiotic resistance marker(s) as BioBrick® standard biological parts (Figure 1a).
  2. Assemble the origin and antibiotic resistance marker via BioBrick® standard assembly (first assembly step, Figure 1b).
  3. Digest the resulting composite part with restriction enzymes XbaI and SpeI (Figure 1c).
  4. Digest the BioBrick® base vector BBa_I51020 with NheI to excise the ampicillin resistance marker BBa_P1006 (Figure 1d).
  5. Since NheI, XbaI and SpeI all generate compatible DNA ends, you can then ligate the composite origin and resistance marker to the linearized base vector (second assembly step).
  6. The ligation product of the composite part and linearized base vector is a functional BioBrick® standard vector (Figure 1e). Although the ligation to make the BioBrick® construction plasmid is not directional, the resulting construction plasmid should be functional regardless.

This plasmid construction approach enables reuse of replication origins and antibiotic resistance markers available from the Registry of Standard Biological Parts. Use of the base vector to construct all BioBrick® standard construction plasmids ensures standardization and uniformity in any resulting BioBrick® vectors.

Entering new BioBrick plasmid backbones into the Registry

Please see the help page on entering new plasmid backbones and their default inserts into the Registry for details.

Construction plasmids built from the BioBrick base vector

Using the approach outlined above, seven new BioBrick construction plasmids were constructed.


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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


Relevant parts

Building new BioBrick vectors: A complete listing of all BioBrick parts that might help you in building a new BioBrick standard construction plasmid.