Help:Plasmid backbones/Construction

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Image:ReshmaShettyPhoto.jpg Image: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 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.
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.


More...
-?-NameDescriptionResistanceRepliconCopy
number		ChassisLength
AWpSB3C5Low to medium copy BioBrick standard vectorCp15A10-12 2738
A  pSB3K5Low to medium copy BioBrick standard vectorKp15A10-12 2936
AWpSB3T5Low to medium copy BioBrick standard vectorTp15A10-12 3252
A  pSB4A5Low copy BioBrick standard vectorApSC101~5 3395
AWpSB4C5Low copy BioBrick standard vectorCpSC101~5 3221
AWpSB4K5Low copy BioBrick standard vectorKpSC101~5 3419
A  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.


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