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Synthetic Biology
Edited by
Robert A. Meyers
Related Titles
The Dictionary of
Schmidt, M. (ed.)
Genomics, Transcriptomics
Synthetic Biology and Proteomics
Industrial and Environmental 5th Edition
Applications
2015
2012 ISBN: 978-3-527-32852-9, also available
ISBN: 978-3-527-33183-3, also available in digital formats
in digital formats
Synthetic Biology
Edited by
Robert A. Meyers
Volume 1
Synthetic Biology
Edited by
Robert A. Meyers
Volume 2
The Editor All books published by Wiley-VCH are carefully
produced. Nevertheless, authors, editors, and pub-
Dr. Robert A. Meyers lisher do not warrant the information contained
Editor-in-Chief in these books, including this book, to be free of
Ramtech Limited errors. Readers are advised to keep in mind that
122, Escalle Lane statements, data, illustrations, procedural details
Larkspur, CA 94939 or other items may inadvertently be inaccurate.
USA
Library of Congress Card No.: applied for
Cover
British Library Cataloguing-in-Publication Data
Design and customization of microcompartments.
A catalogue record for this book is available from
© Martin J. Warren. Detail from figure 12 in
the British Library.
chapter 13 ‘‘Synthetic Biology in Metabolic
Engineering: From Complex Biochemical Bibliographic information published by the
Pathways to Compartmentalized Metabolic Deutsche Nationalbibliothek
Processes – a Vitamin Connection’’. The Deutsche Nationalbibliothek lists this publica-
tion in the Deutsche Nationalbibliografie; detailed
bibliographic data are available on the Internet at
<http://dnb.d-nb.de>.
Contents
Preface IX
Volume 1
4 Interactome 121
Teresa M. Przytycka and Dong-Yeon Cho
5 Microbiomes 153
Ramana Madupu, Yu-Hui Rogers, Doug Rusch, Jason Miller,
Konstantinos Krampis, and Karen E. Nelson
Volume 2
18 The Synthetic Biology Approach to Stem Cells and Regenerative Medicine 541
Boon Chin Heng, and Martin Fussenegger
Index 723
IX
Preface
Synthetic Biology is an emerging field that integrates all biological subsystems with
the objective of engineering, constructing or modifying biological functions, organelles,
cellular structures, simple cells and creating whole novel organisms with designed
properties. This is accomplished by application of engineering principles such as
hierarchical design, modular parts, isolation of unrelated functions and standard
interfaces as well as systems biology and all aspects of molecular biology. Synthetic
Biology extends genetic engineering to focus on whole systems rather than individual
genes and primary gene products, and will ultimately provide diagnostic tools, novel
methods for production of therapeutics and strategies for treatment of diseases. Our
compendium is written for university undergraduates, graduate students, faculty and
investigators at research institutes. Our Board of 11 Nobel Laureates approved our
overall approach and content and our selection of articles was validated and enhanced
by four reviewers from major research institutions. There are 23 peer reviewed articles
at a length of over 700 pages and as such is the largest in depth, up to date treatment
presently available.
The 23 detailed articles organized into six sections: Biological Basis; Modeling;
Modular Parts and Circuits; Synthetic Genomes; Diseases and Therapeutics; and
Chemicals Production. In addition, there is an introductory article entitled Synthetic
Biology. The Biological Basis section defines key areas that support synthetic
biology approaches including Emergence of the First Cells (Protocells), Regulation
of Gene Expression, the Interactome and also Microbiomes; the Modeling section
provides mathematical and computer programming expertise including Dynamics of
Biomolecular Networks, Computer Simulation of the Cell and the SynBioSS Designer
Modeling Suite, which support the following section on Modular Parts and Circuits. This
section covers key practical applications and advances such as Synthetic Gene Networks,
DNA Origami Nanobots, RNAi Synthetic Logic Circuits for Sensing, Information
Processing and Actuation, Synthetic Hybrid Biosensors and Synthetic Biology in
Metabolic Engineering. The Synthetic Genomes section includes articles on Minimal
Gene-Set Machinery; Production of the Mitochondrial Genome and Chromosomal
DNA Segments, and Synthetic Genetic Polymers Functioning to Store and Propagate
Information. As part of the introductory article, Sanjay Vashee, of the J. Craig Venter
Institute, covers the recent work on the formation of a bacterial cell that is controlled by a
chemically synthesized genome. This work was accomplished at the Institute where the
X Preface
author is located. The next two sections cover important end use applications of synthetic
biology. Diseases and Therapeutics covers synthetic biological improvements in use
of stem cells in regenerative medicine and new approaches for vaccine development
utilizing synthetic biology and finally the final section on Chemicals Production includes
synthetic biology approaches for production of diols, biofuels and antibiotics.
We are pleased that many of the major synthetic biology research institutions partic-
ipated in preparation of our book, e.g. the Departments of Biological Engineering, the
Synthetic Biology Center of the Department of Electrical Engineering & Computer Sci-
ence, the MIT Microbiology Program, and the MIT Computational and Systems Biology
Program all of the Massachusetts Institute of Technology; the ETH Zurich, the National
Institutes of Health, and the J. Craig Venter Institute.
Our team hopes that you, the reader, will benefit from our hard work – finding the
content useful in your research and education. We wish to thank our Managing Editor,
Sarah Mellor as well as our Executive Editor, Gregor Cicchetti for both their advice and
hard work in the course of this project.
1
Synthetic Biology:
Implications and Uses*
1 Introduction 2
4 Spatial Regulation 18
4.1 Co-Localization 19
4.2 Compartmentalization 20
7 Concluding Remarks 25
Acknowledgments 25
References 26
*
This chapter has previously been published in: Meyers, R.A. (Ed.) Systems Biology, 2012,
978-3-527-32607-5.
Keywords
Synthetic biology
An effort to construct biological systems, which may include entire biosynthetic
pathways, synthetic organelles and cellular structures, and whole organisms, that
have medical, industrial, and scientific applications. This is achieved via the application
of engineering principles, such as hierarchical design, modular reusable parts, the
isolation of unrelated functions, and standard interfaces.
Synthetic cell
A cell that is controlled solely by a genome that was assembled from chemically
synthesized pieces of DNA.
DNA assembly
The building of larger DNA fragments from smaller DNA fragments.
Circuits
A collection of various modular component parts that responds to an input signal that
is then relayed to produce an output signal.
Compartmentalization
The spatial sequestering of substrates, intermediates, products, enzymes, and activities.
efforts can range in scopes far larger with recent advances in the field, such
than the traditional genetic engineering of bioengineering projects will become faster
genes, to include the engineering of entire to develop, easier to operate, and also much
biosynthetic pathways complete with the more ambitious.
regulation of the genes in that pathway During recent years, Synthetic Biology
[3, 4], synthetic organelles and cellular has progressed in a manner which is very
structures [5], whole organisms [6–9], and different from those of other engineering
even ecosystems [10–13]. Synthetic Biol- disciplines. This is because, unlike archi-
ogy has the ambition to apply classical tecture or software engineering, there is
engineering principles such as hierarchi- already a reservoir of highly sophisticated
cal design, modular reusable parts, the and complex functional parts to be found
isolation of unrelated functions, and stan- in Nature, and consequently most efforts
dard interfaces. The empirical fields that in Synthetic Biology have been focused on
correlate to Synthetic Biology are Systems harnessing that natural resource base. In
Biology, Genetics, and Molecular Biology. general, two basic approaches have been
Synthetic Biology is not a new field, but undertaken to achieve this feat. The first
rather extends back into prehistory. For approach has been to engineer natural
example, it has been determined that the organisms so as to incorporate recombi-
process of engineering maize – a highly nant pathways and other such desirable
optimized domestic agricultural crop plant attributes. This method has the advantage
– from the wild grass teosinte began of not requiring the capability to build –
over 9000 years ago [14]. The method nor require an understanding of – mas-
used by the pre-Columbian cultivators sive biological systems such as genomes
of teosinte was simple artificial selection and metabolisms. However, it does have
which, as such, is very slow. However, the disadvantage of being undefined; that
with the discovery of laws of inheritance is, whilst certain genes of the organism
and natural selection [15–17], and the might be the result of human intervention,
suggestion that DNA was the chemical most of the genome remains wild-type,
medium of inheritance [18], the scene and is neither subject to human control
was set to engineer a living system in nor necessarily operating within the limits
a far more direct and rapid manner. of human knowledge.
A prominent example of this is the The alternate approach is to use func-
Dupont Escherichia coli strain used for the tional components, originally ‘‘mined’’
production of 1,3 propanediol, in which from Nature, such as promoters, ap-
case an entire biosynthetic pathway has tamers, protein–protein interaction do-
been added to E. coli, and the metabolism mains, terminators, or ribosome-binding
of the bacterium substantially altered sites. These functional components can be
to allow for a majority of the carbon cataloged and then used to compose larger
feedstock (glycerol) to be converted into defined constructions of genes, pathways,
the economically valuable chemical 1,3 and even whole genomes. Because all
propanediol [6, 8, 9]. This feat, which the components of a synthetic biological
was begun prior to the development of system that are constructed in such an
most of the Synthetic Biology techniques approach have precisely defined proper-
reviewed in this chapter, took many years ties, a high degree of predictive control
and substantial investment to achieve. Yet, over the final product is afforded. Indeed,
4 Synthetic Biology: Implications and Uses
this more defined approach has become combination of recombinant DNA cloning
synonymous with advances in Synthetic techniques with the PCR allowed a much
Biology. greater exploitation of natural biological
Historically, one of the main limitations components, such as thermostable DNA
in following this defined approach relates polymerases, and this in turn made the
to the knowledge of these natural systems PCR more robust and practical. Today, the
that serve as a source of parts. As biology PCR has become an indispensable tool for
has been characterized, both new com- biology. Thus, knowledge of natural sys-
ponents for synthetic biology – and also tems has led to an improved technology
new tools to utilize those components – for exploiting those systems, which has
have become available. In turn, a new in turn provided an improved knowledge
Synthetic Biology capability has driven of biological systems in a double feed-
greater advances in the understanding of back loop, thus improving both scientific
biology. This has been most obvious in understanding and technological capabili-
the development of tools for the synthe- ties. As shown in Fig. 1, as knowledge of
sis and manipulation of DNA, the first of the fundamental principles of biology have
which were developed via the discovery continued to grow, it has been possible to
of restriction endonucleases, DNA ligases, take a more defined engineering approach.
and the creation of recombinant DNA In the past, advances in synthetic biol-
molecules [19–24]. These tools allowed ogy have been bounded by the capacity
the development of the recombinant DNA to assemble and modify DNA, as well
cloning and expression techniques that ul- as knowledge of biological parts and cir-
timately made possible the exploitation of cuits that such DNA might encode. Cor-
enzymes with desired activities and prop- respondingly, these two areas are directly
erties. Notably, the development of the addressed in the following sections, with
polymerase chain reaction (PCR) greatly details of synthetic biological pathways,
increased the ability to amplify and ma- synthetic genomes, synthetic organelles,
nipulate DNA [25–27]. The subsequent and even synthetic organisms provided as
Progress in Synthetic Biology is defined by the shifting of life-manipulation
from the undefined to defined products and techniques.
(fast, powerful, but (slow, limited in scope,
requires substantial but requires little or no
biological knowledge) biological knowledge)
Wholly Wholly
Defined Undefined
Custom Custom
Biological Biological
Systems Systems
examples of the capabilities that Synthetic restriction enzyme-based methods are still
Biology has already begun to deliver. commonly in use [28, 29], these methods
These new capabilities create, in turn, are gradually being superseded by the de-
new societal challenges, and these are also velopment of very rapid, more robust and
discussed. less limited DNA assembly techniques.
For example, although BioBricks were
originally designed to be assembled with
2 a restriction enzyme/ligation method [30],
DNA Assembly and Modification more recently an in vitro homologous
recombination was adapted to increase
As discussed above, one way to view the flexibility and speed of BioBrick
Synthetic Biology is as an engineering assembly [31].
discipline aimed at manipulating cellu- The starting materials for DNA se-
lar systems to produce a de novo-designed quence construction may include chem-
function that does not exist in the nat- ically synthesized DNA oligonucleotides
ural organism. As with other engineer- (oligos), natural DNA fragments, PCR
ing fields, however, Synthetic Biology is products, or a combination of all three
dependent on the tools and techniques sources. Defined short single-stranded oli-
available. gos have been commercially available as a
Organisms carry out a variety of reac- commodity for many years, usually for use
tions aimed at their self-sustenance and as primers in PCR, mutagenesis, and se-
self-replication, with such reactions being quencing reactions. Since chemically syn-
carried out by the proteins and RNAs en- thesized DNA oligos are of a user-defined
coded in the organism’s genome. As the sequence, this allows for a nucleotide level
sequence of the DNA directs production control of gene sequences and even entire
of the proteins and RNAs of an organism, genome sequences – a firm requirement
control of the cellular DNA therefore al- when designing new functions in organ-
lows for an ability to direct the functions isms. The idea of synthesizing genes from
of a cell. Based on this principle, many DNA oligos is not new; previously, oligos
of the basic tools utilized in Synthetic have been used to synthesize genes such
Biology are aimed at producing defined as the alanine tRNA from yeast [32] and
sequences of DNA molecules, and eas- the human leukocyte interferon gene [33].
ily manipulating the DNA content of an Likewise, the gene encoding a mammalian
organism. The DNA molecules necessary hormone, somatostatin, was synthesized
for Synthetic Biology purposes can vary and expressed in E. coli [34]. It is only
greatly in length, from individual DNA recently that the lower costs of oligonu-
parts, genes and plasmids (containing tens cleotide synthesis and DNA sequencing
to thousands of base pairs) to biosynthetic have been combined to allow the devel-
pathways and genetic circuits (thousands opment of more cost-effective and rapid
to millions of base pairs) to synthesizing methods for assembling groups of oligos
whole genomes (viral and bacterial). into synthetic pieces of DNA or genes
For many years, gene cloning and DNA [35–39]. Today, several commercial gene
assembly were dominated by the use synthesis companies exist that are able to
of restriction endonucleases and DNA produce custom genes/DNA at an accessi-
ligases [19–24]. While some well-designed ble cost per base pair, although such costs
6 Synthetic Biology: Implications and Uses
can quickly become prohibitive if numer- utilizing the natural homologous recom-
ous different DNAs are required. The cost bination activity of an organism, such as S.
prohibition of large-scale gene synthesis cerevisiae (yeast) and B. subtilis [7, 40–44].
can, in part, be overcome by utilizing nat- Occasionally, a method will include an
ural DNA fragments and PCR products in vitro step to perform a partial reaction
in the DNA assembly for those sections (DNA chewback/DNA annealing/DNA ex-
of DNA that do not need to be created tension), and an in vivo step to complete
synthetically. the reaction (DNA repair) [38, 45, 46].
Typically, the process of constructing The various in vitro homologous re-
DNA is hierarchical (Figs 2 and 3). combination methods used to assemble
Briefly, groups of smaller DNAs (single- or double-stranded DNA or single-stranded
double-stranded) are mixed and assembled oligos share the same general mechanism
into larger DNA pieces. Figure 2 shows (Fig. 2). The nucleotides are first removed
double-stranded DNAs being assembled from one strand of the overlapping ends of
into a larger construct, but the process can the adjacent double-stranded DNAs, thus
begin with single-stranded oligos as the creating single-stranded ends of the DNA
substrates. These larger pieces (subassem- (Step 1). This process is analogous to a
blies) are then grouped and assembled. restriction enzyme digestion creating com-
These steps can be repeated until the fi- plementary sticky ends of DNA, except
nal full-length DNA construct is obtained, that the single stranded ends are typically
whether it is a gene or genome. The DNA 20–60 nucleotides long. Depending on the
pieces to be assembled must have homol- method used, the nucleotides can be re-
ogous overlapping ends, the overlaps be- moved by applying an exonuclease activity
ing important because the DNA assembly from either the 5 or 3 ends of the DNA
techniques utilize homologous recombi- [37, 38]. The creation of single-stranded
nation. For example, if three pieces of DNA overhangs (Step 1) is not necessary if oli-
(A, B, and C) are to be assembled into a sin- gos are used as the starting material for
gle DNA molecule, then one end of piece A DNA assembly, because the oligos are al-
must have an overlap with piece B, and the ready single-stranded. The single-stranded
other end of B must overlap piece C. This DNA overhangs are complementary to
configuration will result in a linear DNA each other on adjacent molecules, and thus
molecule, A–B–C. In order to generate a are able to anneal (Step 2). If the ends of
circle from these pieces, the end of C must the molecule being constructed are com-
overlap piece A. The assembly of DNA plementary, then the final construct will
into a circle is most often achieved with be circular (this is the normal method
a DNA piece which contains sequences used when DNA is being assembled into
that enable the final construct to be cloned a cloning or expression vector). The final
into a desired host (e.g., E. coli, Saccha- step to the reaction is repair of the DNA.
romyces cerevisiae or Bacillus subtilis) [37, 38, In the in vitro reaction, a DNA polymerase
40–42]. The DNA homologous recombina- is used to fill the gaps, while a DNA ligase
tion reaction can be carried out completely seals the nicks so as to create the larger as-
in vitro in one step, either with an enzyme sembled DNA molecule. The DNA repair
mix [37] or by using the PCR [35]. The activity of E. coli can be utilized to com-
reaction can also be performed in vivo by plete the reaction after the annealing step
Synthetic Biology: Implications and Uses 7
A B C
5′ 3′ 5′ 3′ 5′ 3′
3′ 5′ 3′ 5′ 3′ 5′
T5 exonuclease (5′)
1. Recessing of DNA to create
single-stranded complementary DNA Exo III exonuclease (3′)
either at 5′ or 3′ ends. T4 DNA polymerase (-) nucleotides (3′)
5′ 3′ 5′ 3′ 5′ 3′
3′ 5′ 3′ 5′ 3′ 5′
2. Annealing of single-stranded
complementary ends.
3′ 5′ 3′ 5′
5′ 3′ No homology
3′ 5′ 3′ 5′ 3′ 5′ at ends.
(Or)
3′ 5′ 3′ 5′
5′ 3′ Homology
3′ 5′ 3′ 5′ 3′ 5′ at ends.
Phusion DNA polymerase
3. DNA repair. Taq DNA polymerase
Fill in of gaps and ligation of nicks. Taq ligase
In vitro repair (circular assemblies only)
5′ 3′
3′ 5′
Linear assembly product
Title: Kertomuksia I
Language: Finnish
KERTOMUKSIA I
Kirj.
Anton Tshehov
Osakeyhtiö Kauppakirjapaino.
SISÄLLYS:
Öljytikku.
Yksi monista.
Virkateitä.
Nopeaa apua.
Hermostunut.
Käräjiin menossa.
Mokomaa väkeä.
Haudalla.
Se ei ollutkaan hän.
Pieni erehdys.
Toimen miehiä.
Onnenmyyrä.
Anjuta.
Molempi parempi.
Ei kelpaa.
Tupakan vahingollisuudesta.
Matkan varrella.
ANTON PAWLOWITSH TSHEHOW.
Suomentaja.
KERTOMUKSIA
ÖLJYTIKKU.
(Rikosjuttu).
I.
— Keltainen, purjekankaasta.
— Ja sitte?
— Missä te heräsitte?
II.
— Mitäs te nyt?
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