Haynes:BioBrick Method short

Assembly of BioBrick Parts: an Overview

By Karmella Haynes, 2012

If you are new to molecular biology, please refer to a general biology reference (college biology text book, Wikipedia, etc.) or click the "ref" link for more details on the terms in bold print.

A BioBrick is a standardized piece of DNA that is flanked by a set of restriction enzyme cut sites (ref), e.g., EcoRI (E), NotI (N), XbaI (X) upstream (left); SpeI, NotI, PstI downstream (right). What makes the DNA a "standard part" is that the users within a community agree to flank their DNA fragments with the same sites, and they make sure that the cut sites do not appear within their DNA parts, so that parts can be shared freely without the time-consuming process of figuring out a new strategy every time a user wants to assemble a synthetic gene. The standard shown below is RFC23, or the "Silver Standard" developed by Pam Silver's lab at Harvard:

gaattc gcggccgc a tctaga [BioBrick Part] actagt a gcggccgctgcag
  EcoRI   NotI       XbaI                   SpeI     NotI   PstI  
BioBrick Prefix                       BioBrick Suffix

Note: The NotI and PstI cut sites overlap at the underlined “c.”

The BioBrick itself must not contain any of the standard restriction sites (internally), or else it will be cut apart during the assembly process. Basic BioBrick assembly uses one of the two strategies:

Ligating a “front insert” in a “front vector” to make A+B

Ligating a “back insert” in a “back vector” to make the same A+B construct