BioBrick Assembly Standards and Techniques and Associated Software Tools - DNA Cloning and Assembly Methods - page 2

Fig. 1 BioBrick standard assembly prefix and suffix sequences

domains possible. Stability and degradation of the fusion proteins is
also an issue that has led to new proposed assembly standards. In the
following subchapters, the five assembly standards supported by
the Registry of Standard Biological Parts, in addition to three of the
most used assembly techniques will be explained.

1.1 Assembly
Standards

BBF RFC 10, called BioBrick standard assembly [ 3 ], was proposed
by Tom Knight in May 2007 and is the BioBrick standard that is
most commonly used. Most BioBricks listed on the website of the
Registry of Standard Biological Parts ( http://partsregistry.org/
Main_Page ) is compatible with this assembly method.
BioBricks following the BBF RFC 10 assembly standards have
one standardized suffix, but the prefix may appear in one of the
two ways, depending on whether or not the BioBrick in question
is a protein-coding sequence or not. The two prefixes and the suf-
fix used by bricks that are compatible with BioBrick standard
assembly are given in Fig. 1 .
In both prefixes, restriction sites for EcoRI (GAATTC) and
NotI (GCGGCCGC) are present. Additionally, for noncoding
BioBrick parts the prefix contains an XbaI (TCTAGA) restriction
site. In the case of the prefix used for protein-coding parts, the
XbaI restriction site appears only when the prefix sequence is fused
with a protein-coding sequence starting with ATG. In the suffix,
restriction sites for SpeI (ACTAGT), NotI (GCGGCCGC), and
PstI (CTGCAG) are present.
The most common way of joining together two BioBrick parts
following the BBF RFC 10 assembly standard is to digest the first
BioBrick with EcoRI and SpeI, creating an insert, and the second
BioBrick with EcoRI and XbaI, creating a backbone with an open-
ing in front of the second BioBrick part. When the two digested
fragments are joined together, the sticky ends created by EcoRI on
the two DNA molecules will be ligated back together to restore
the EcoRI restriction site. Both XbaI and SpeI create compatible
overhangs, so the sticky ends created by digesting with XbaI and
SpeI can be ligated together. When this is performed, the ligated
sequence will be a mix of the two restriction sites, and the new

1.1.1 BioBrick
Standard Assembly