Plasmid backbones/Construction

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Plasmid backbones/Construction
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You may find that there does not already exist a BioBrick® plasmid backbone that fulfills your requirements. You may need a different antibiotic resistance, a different copy number, or something else.

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.

For convenience, we also describe the approach here. Or, you may wish to jump directly to the catalog of BioBrick parts for building BioBrick vectors.

Overview

Reshma's method for building new BioBrick® vectors relies on building BioBrick® vectors entirely from BioBrick® parts. To make the process of building BioBrick® vectors easier, Reshma designed a BioBrick® base vector (BBa_I51020). Using the based vector, you can construct a new BioBrick® vector from (almost) any replication origin and antibiotic resistance marker, as long as the origin and antibiotic resistance marker are themselves BioBrick® parts. In a nutshell, the method allows you to assembly any replication origin and antibiotic resistance marker together into the BioBrick base vector to construct a new, functional BioBrick vector. The base vector already has many of the general plasmid features that you might need in a BioBrick® vector, so use of the base vector ensures standardization and uniformity of any resulting BioBrick® vectors. (A complete list of all design features of the BioBrick® base vector is available.) BBa I51020schematic.png

Method

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 plasmid inserts into the Registry for details.

BioBrick plasmid parts

Browse all BioBrick plasmid parts.