Digestion Guide

Schematic showing cut sites (red arrows) on part and destination vectors, reproduced in part from Torella et al. 2014, NAR

Which Sites to Digest?

Once DNA sequences of interest are cloned into the part vectors (e.g. by restriction cloning into BioBrick sites in the MCS), UNS-flanked parts can be generated by restriction digestion of sites flanking the UNSes (schematic, right). For all but the most 3' part to be assembled, this involves cutting around the U_N and U_N+1 sites. The most 3' part should instead be digested around the U_N and U_X sites. This mixture of parts can then be Gibson assembled with one another and with the U₁ and U_X sites in each destination vector.

For example, let's say we've cloned genes A, B and C into part vectors U₁U₂, U₂U₃ and U₃U₄. In a three-part assembly, the U₁U₂ part vector would be cut around the U₁ and U₂ sites and the U₂U₃ part vector would be cut around the U₂ and U₃ sites. The last part, however (U₃U₄ vector) would be cut around the U₃ and U_X sites. This would yield the following linear DNAs:

[U₁-A-U₂] [U₂-B-U₃] [U₃-C-U₄-U_X]

Digesting the destination vector as shown in the schematic (top right) will give:

[U_X-Backbone-U₁]

Which can be circularized with the other parts via Gibson assembly.

Because the last (most 3') part in the assembly must be cut with different restriction enzymes than the other parts, it is important to plan the restriction digests carefully to ensure proper assembly. A table of restriction sites that cut at each site on each of the available part and destination vectors is given below: