Three antibiotic assembly (3A assembly) is a method for assembling two BioBricks using restriction enzyme cloning. Three different antibiotic resistances allow for efficient selection for correct assemblies through antibiotics. 3A assembly uses the restriction sites on the prefix and suffix to assemble the two parts.

Effective antibiotic selection eliminates the need for gel purification and colony PCR of the resulting colonies. In theory, about 97% of the colonies should be the desired assembly.


For a successful assembly you need two parts and a backbone with different antibiotic resistance genes (symbolized by yellow arrows in figure 1).

Figure 1: 3A assembly (attribution

  • Restriction digests

    • The left part sample is cut out with EcoRI (E) and SpeI (S).

    • The right part sample is cut out with XbaI (X) and PstI (P).

    • The plasmid backbone is cut with EcoRI (E) and PstI (P). In the example of figure 1 a linearized plasmid backbone is used. Alternatively a plasmid containing a ccdB gene can be used.

  • All 3 restriction digests are heated to heat kill all of the restriction enzymes.

  • Ligation: An equimolar quantity of all 3 restriction digest products are combined in a ligation reaction. The desired result is the left part sample's SpeI overhang ligated with the right part sample's XbaI overhang resulting in a scar that cannot be cut with any of our enzymes. The new composite part sample is ligated into the construction plasmid backbone at the EcoRI and PstI sites.

  • When the ligation is transformed into cells and grown on plates with antibiotic C, only colonies with the correct construction survive.