C H A P T E R T H I R T E E N
Assembly of BioBrick Standard
Biological Parts Using Three
Antibiotic Assembly
25
* Depa
{ Com
Camb
1 CurreMethods in Enzymology, Volume 498 # 2011 Elsevier Inc.
ISSN 0076-6879, DOI: 10.1016/B978-0-12-385120-8.00013-9 All rights reserved.
311oped BioBrick assembly standard 10 to enable the construction of systems from
standardized genetic parts. The BioBrick standard underpins the distributed
efforts by the synthetic biology research community to develop a collection of
more than 6000 standard genetic parts available from the Registry of Standard
Biological Parts. Here, we describe the three antibiotic assembly method for
physical composition of BioBrick parts and provide step-by-step protocols.
rtment of Biological Engineering,Massachusetts Institute ofTechnology,Cambridge,Massachusetts,USA
puter Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology,
ridge, Massachusetts, USA
nt address: Ginkgo BioWorks, Inc., Boston, Massachusetts, USAbiological systems easier and more reliable. In support of this goal, we devel-
An underlying goal of synthetic biology is to make the process of engineering
AbstractRefe
knowledgments
rences 3
3258. C
Aconclusions 325
7. Preparation of Chemically Competent Cells 323
6. Available BioBrick Destination Vectors 323Improve 3A Assembly 321
5. Preparation of Linearized Destination Vector by PCR to4. Verification of Correct Assembly of BioBrick Standard
Biological Parts 320Reshma Shetty,*,1 Meagan Lizarazo,† Randy Rettberg,*,† and
Thomas F. Knight†,1
Contents
1. Introduction 312
2. Construction of New BioBrick Standard Biological Parts 316
3. 3A Assembly of BioBrick Standard Biological Parts 318

and Rettberg, 2009). For brevity, here we use the term BioBrick parts to refer
to parts that adhere to BioBrick assembly standard 10.
In
stand
pairw
312 Reshma Shetty et al.addition to supporting the distributed development of a collection of
ard biological parts, the BioBrick standard also enables the iterative and
ise hierarchical assembly of genetic parts into systems containing manyThe method relies on a combination of positive and negative selection to
eliminate time- and labor-intensive steps such as column cleanup and agarose
gel purification of DNA during part assembly.
1. Introduction
A fundamental goal of synthetic biology is to make the process of
designing, building, and testing biological systems easier and more reliable.
Drawing inspiration from other engineering fields, we have been working
to develop methods that support the design and construction of multicom-
ponent, synthetic biological systems from standardized biological parts. In
2002, Knight proposed and implemented the BioBrick standard for assem-
bly of standard genetic parts (Knight, 2003). Parts that have been refined to
adhere to the BioBrick assembly standard are called BioBrick standard
biological parts. The key innovation of the BioBrick standard is that
assembly of any two BioBrick parts yields a composite object that is itself
a BioBrick part that can be further combined with any other BioBrick parts.
The idempotent nature of the BioBrick standard has helped to support the
distributed development of a collection of over 6000 standard biological
parts in the Registry of Standard Biological Parts (http://partsregistry.org).
Parts in the Registry are cataloged, describing and conveying units of
biological function rather than simply arbitrary DNA sequences. Participants
in the international Genetically Engineered Machines competition (iGEM,
http://igem.org) as well as many academic laboratories have made use of the
BioBrick assembly standard in construction of genetic parts, devices, and
systems (Afonso et al., 2010; Agapakis et al., 2010; Ajo-Franklin et al., 2007;
Canton et al., 2008; Du et al., 2009; Gru¨nberg et al., 2010; Haynes et al., 2008;
Huang et al., 2010; Kelly et al., 2009; Levskaya et al., 2005; Shetty et al., 2008;
Tabor et al., 2009). Several additional physical composition standards have
been proposed that extend or build upon the BioBrick assembly standard
(Anderson et al., 2010; Ellison et al., 2009; Gru¨nberg et al., 2009; Knight, 2008;
Peisajovich et al., 2009; Phillips and Silver, 2006). The BioBricks Foundation
(BBF) nowmanages an open standards setting process for the synthetic biology
community via the submission and publication of requests for comment
(RFCs). In accordance with BBF RFC 29, the BioBrick assembly standard
is now referred to as BioBrick assembly standard 10 in theRegistry of Standard
Biological Parts, and other assembly standards are similarly numbered (Shetty