Synthetic Biology and Gene Cloning
NJ Patron, The Sainsbury Laboratory, Norfolk, UK
2017 Elsevier Ltd. All rights reserved.
Glossary
Archeaplastida A group of plastid-containing eukaryotes
comprising red algae, green algae, land plants, and
glaucophytes.
Binary plasmid vector Plasmids that contain replication
origins for both Escherichia coli, an organism where genetic
modication is easy, and Agrobacterium tumefaciens. The
genes that facilitate transfer of the T-DNA are housed on
a separate DNA molecule.
BioBricks BioBrick parts are DNA sequences that conform
to a restriction enzymemediated assembly standard.
DNA Ligase An enzyme that can catalyze the joining
together of two DNA molecules.
Exonuclease Enzymes that cleave nucleotides one at a time
from the ends of polynucleotide chains.
Gene synthesis The chemical synthesis of a DNA molecule
of any sequence.
Gibson assembly A DNA assembly method that joins
multiple DNA fragments in a single reaction aided by three
enzymes.
Left/right border (LB/RB) These are 25 base pair imperfect
repeats that ank the T-DNA region in tumor-inducing (Ti)
plasmids and binary plasmid vectors.
Origin of replication Also called the replication origin, is
the region of a DNA molecule at which replication is
initiated in vivo.
Overlap-dependent cloning DNA assembly methods that
rely on sequence identity at the ends of multiple DNA
fragments to enable their assembly into a single molecule.
Parallel assembly The assembly of multiple fragments of
DNA in a single reaction.
Polymerase chain reaction (PCR) An in vitro technology
that replicates a specic DNA fragment generating
thousands to millions of copies.
Abbreviations
dCTP Deoxy-cytidine triphosphate
dNTPs Deoxy-nucleoside triphosphates
LB Left border
PCR Polymerase chain reaction
Introduction
Molecular cloning is the process in which DNA molecules are
assembled and replicated in a host organism. This is usually
done with the aim of producing sufcient quantities of any
specic DNA sequence to allow for experimentation. In plant
112 Encyclopedia of Applied Plant Sciences, 2nd edition, Volume 2
Plasmid A DNA molecule physically separated from the
chromosomal DNA that can replicate independently. Most
commonly found in bacteria as circular, double-stranded
DNA molecules.
Plastid A double-membrane-bound organelle found in the
cells of plants and algae, thought to have originated from
an endosymbiotic cyanobacteria 1.5 billion years ago.
Scarless cloning The joining of two or more fragments of
DNA in such a way that no additional, unwanted
nucleotides are introduced between the sequences of
interest.
Selectable marker gene A selectable marker is a gene
introduced into a cell to confer a trait suitable for articial
selection, usually of an externally supplied chemical agent
such as an antibiotic or herbicide.
Sequence and ligation independent cloning (SLIC) A
DNA assembly method that can be used to join DNA
fragments into a single molecule, regardless of sequence,
without the aid of a DNA ligase.
Shuttle chassis An organism that carries, or shuttles, a DNA
molecule between the organism in which it was
constructed and the destination chassis or organism.
T-DNA The region of a tumor-inducing (Ti) or binary
plasmid anked by left and right border sequences and
transferred to the plant genome during Agrobacterium-
mediated transfection.
Type II restriction endonucleases Enzymes that cut DNA
at dened positions close to or within their, usually
palindromic, recognition sequences.
Type IIS restriction endonucleases Enzymes that cut DNA
at dened positions up- or downstream of their non-
palindromic recognition sequences.
RB Right border
SLIC Sequence and ligation independent cloning
T-DNA Transfer-DNA
biology the cloned DNA is ultimately transferred to a plant
cell. The aim may be to elucidate the function of the cloned
DNA, to reprogram the plant to perform new functions
encoded by the cloned DNA, or to use the plant cell as
a production system or factory for a protein or metabolite
of interest.
http://dx.doi.org/10.1016/B978-0-12-394807-6.00132-5
 Biotechnology j Synthetic Biology and Gene Cloning 113

Until recently the bottlenecks of cloning plant genes were the
identication and isolation of target genes and the assembly of
multiple sequences into large and complex constructs. Over
the past decade the invention and application of next generation
sequencing technologies have reduced the speed and cost of
genome sequencing, overcoming many of the barriers to the
identication of novel DNA sequences. Even more recently,
DNA assembly technologies have been revolutionized with
several robust technologies for the parallel assembly of multiple
DNA molecules being widely adopted. These technologies have
been critical to the burgeoning eld of synthetic biology.
Synthetic biology is an approach to engineering biology
that augments molecular biology and automated sequencing
technologies with new foundational technologies and princi-
ples successfully employed in mechanical and electronic engi-
neering including the automation of design and construction;
the standardization, modularization, and characterization of
components, tools, and assembly methods; and the establish-
ment of abstraction hierarchies to allow the engineering of
complex systems. The principles and core technologies of
synthetic biology are summarized in Figure 1.
With the advent of cheap gene synthesis services where
bespoke sequences can be ordered at increasingly low cost,
a growing number of laboratories no longer bother to isolate
and clone specic sequences, particularly if the organism is
not readily available. Instead, data from genome sequences
and transcriptional studies are mined and sequences of interest
are ordered ready for assembly into desirable congurations. In
this article the features of plasmid backbones in which
sequences are assembled for delivery to plant cells are described
along with several methods for the assembly of DNA molecules
into such plasmids.
Plasmid Vectors for Plant Biology
Plasmid backbones have several specic features, the most
important of which are (1) that they contain the correct
features for replication at the desired copy number in the
host cell(s) of interest, (2) that they contain the sequences
required for delivery to host, and (3) that they contain or
are free from any specic sequences required for or that
interfere with the chosen DNA assembly technique. Several
technologies for the transfer of DNA and proteins to plant
cells have been in use since the 1980s. These include the
use of shuttle chassis, most commonly, Agrobacterium tume-
faciens, to deliver DNA assembled in other bacteria to plant
cells as well as the direct application of DNA to plant cells.
Plasmids for Agrobacterium-Mediated DNA Delivery
A. tumefaciens is an alphaproteobacteria with the ability to trans-
fer a region of a large tumor-inducing (Ti) or rhizogenic (Ri)
plasmid to plant nuclear genomes. In the 1980s, this species

Figure 1 The core principles and technologies of synthetic biology are (a) the use of polymerase chain reaction (PCR) to amplify specic DNA
molecules into sufcient quantities for experimentation and engineering, (b) the use of recombinant DNA technologies to assemble specic DNA
sequences in novel combinations, (c) chemical synthesis of oligonucleotides and automated assembly into bespoke DNA molecules, (d) the use of
abstraction hierarchies to enable bioengineers and designers to design molecular networks without reading DNA sequences, but instead to assemble
genetic elements known to perform specic functions, (e) the automated assembly of multiple characterized standard DNA parts into genetic circuits,
and (f) the application of automated sequencing technologies to verify the correct assembly of DNA molecules and their integration and effectiveness
in biological systems.
114 Biotechnology j Synthetic Biology and Gene Cloning

was repurposed for the genetic modication of plant genomes
by the removal of the genes that cause pathogenic symptoms.
The region of the Ti or Ri plasmid transferred to the plant is
known as the transfer region or transfer DNA (T-DNA). The pro-
cessing of the T-DNA from the plasmid, its export from the
bacterium, and its entry into the plant nuclear genome is medi-
ated by virulence (vir) proteins, which interact with several plant
proteins. T-DNAs are dened by homologous, 25-base-pair
sequences in direct repeats known as the left (LB) and right
(RB) borders. A so-called overdrive sequence close to the RB
is also involved in enhancing the transmission of the T-DNA.
Ti plasmids are very large and replacing the T-DNA with genes
of interest used to involve several complex steps. To simplify this
process two strategies were employed: The rst involved either
conjugation or transformation of a Ti plasmid to introduce
new sequences in cis with the virulence genes. The second, and
by far the most widely used system, is to clone DNA sequences
of interest between the LB and RB on a small plasmid, separate
to the plasmid on which the vir genes are housed. This is called
the binary vector system. Apart from the T-DNA region, the small
binary plasmid vectors also contain a high-copy origin of replica-
tion to facilitate cloning in fast-growing Escherichia coli as well as
an origin of replication for A. tumefaciens. Assembled plasmids
are transferred to strains of A. tumefaciens that carry vir genes,
the products of which will mediate the insertion of the T-DNA
into the plant genome (Figure 2(a)).
Plasmids for Direct-Delivery Methods
DNA can also be delivered directly to plant cells without the aid
of a shuttle chassis. In order to penetrate the plasma and
nuclear membranes, DNA must either be propelled though
the cell wall by force in a process known as biolistic or
gene-gun-mediated DNA delivery. Alternatively, the cell wall
is removed enzymatically, releasing wall-free plant cells known
as protoplasts from plant tissues. Many protoplasts are able to
take up DNA through their plasma membrane and, depending
on the species and tissue from which the cells were isolated, can
be induced to repair their cell wall and to divide forming new
tissue from which whole plants can be regenerated.
Large quantities of DNA are required for direct DNA-
delivery methods but, other than a high-copy origin of replica-
tion to facilitate the production of large amounts of plasmid
DNA, no other features are necessary for plasmids used for
the direct delivery of DNA to nuclear genomes (Figure 2(b)).
It fact, it is possible to release the cloned DNA from the plasmid
backbone for delivery in order to avoid integration of the
plasmid backbone along with the genes of interest.
Delivery to the plastid genome is desirable either for the
express purpose of engineering the plastid genome or for the
production of proteins or metabolites within this cellular
compartment. As each plant cell, particularly those found in
green tissues, may contain a large number of plastids, engi-
neering the plastid genome can enable the production of
sizable quantities of gene products. Plastid engineering for
the production of vaccines and other medicinal compounds
has been particularly successful.
Plastids are thought to be of prokaryotic origin, the result
of the retention and reduction to an organelle of an ancient
cyanobacterial endosymbiont in a single-celled eukaryotic
ancestor of Archeaplastida. Plant plastids retain homologous
recombination as the preferred DNA repair pathway, and this
can be utilized for the integration of foreign DNA into their
genomes. For delivery to plastid genomes, DNA sequences of
interest are cloned into plasmids anked by regions of
homology to the target plastid. After delivery to the plant tissue,
usually by a direct-delivery method, the homologous regions
recombine with the plastid integrating the foreign DNA
between these borders (Figure 2(c)).
DNA Assembly Methods
Single Gene Cloning
The vast majority of transgenic plants produced in the past
three decades have had just a single recombinant gene, together

Figure 2 Plasmid backbones require several specic features depending on their use. (a) Plasmids used for Agrobacterium-mediated transfer of
DNA to plant nuclear genomes must contain origins of replication for Escherichia coli (ori Ec), an organism in which genetic modication is easy, and
for Agrobacterium tumefaciens (ori At). They must also contain Left and Right Border (LB and RB) sequences to dene the T-DNA region. The pro-
cessing and transfer of the T-DNA to the plant cell is enabled by virulence (vir) proteins, coded on a separate DNA molecule in the A. tumefaciens
cell, (b) Plasmids used to replicate DNA for direct-delivery methods do not require any special features. A high-copy origin of replication for E. coli
(ori Ec) is desirable as large quantities of DNA are required for these methods, (c) For delivery to plastid (p) genomes DNA sequences of interest are
cloned between regions of DNA with homology to the plastid genome. After delivery to the plant these homologous sequences recombine with the
plastid DNA, integrating the foreign DNA between them.
 Biotechnology j Synthetic Biology and Gene Cloning 115

with a selectable marker gene, transferred to their genome.
Selectable marker genes generally confer resistance to a selective
agent such as an antibiotic or herbicide. They allow plant cells
in which the transferred DNA has integrated into the nuclear
genome to regenerate while non-transformed cells will either
senesce or will be unable to continue normal growth and divi-
sion when the selective agent is applied.
Prior to the establishment of parallel DNA assembly
methods that allowed multiple fragments of DNA to be assem-
bled in a single step with high efciency, several plasmid vector
series were developed with plant selectable marker genes
inserted into the backbone alongside a region of DNA with
features that enabled the facile cloning of any gene or coding
sequence of interest (Figure 3). The simplest sequence feature
for the acceptance of single fragments of DNA is a multiple
cloning site, a region of DNA with multiple recognition
sequences for relatively rare Type II restriction endonucleases
into which the sequence of interest can be cloned. Another
widely used cloning method is Gateway Cloning. This
method efciently transfers DNA anked by site-specic
recombination sites known as att sites using a proprietary set
of enzyme mixes known as clonases (Figure 3(b)). Genes of
interest are cloned into so-called ENTRY plasmids anked
by attR sites. The ENTRY clone is then incubated with the desti-
nation plasmid that contains attL sites and the clonase transfers
the fragment of interest by recombining the attR and attL sites
(Figure 3(b)). Any plasmid vector can be converted for Gate-
way Cloning by the addition of the accepting attL sites,
usually anking a negative cloning selection gene ccdB. This
prevents bacteria transformed with plasmids in which the
sequence of interest has not replaced the ccdB gene from form-
ing colonies, allowing for easy selection of positive clones.
Parallel and Standardized DNA Assembly Methods
In recent years the drive to engineer complex traits has increas-
ingly required the assembly of large multigene pathways. This
has driven innovation in DNA assembly technologies. New
assembly technologies can generally be classied into two
groups. Those that produce scarless assemblies with no
unwanted nucleotide bases between DNA fragments and those
that add anking sequences containing specic restriction
enzyme recognition sites to the start and end of each DNA frag-
ment to be assembled, standardizing the assembly protocol.
To date, Gibson Assembly and Sequence and Ligation Inde-
pendent Cloning (SLIC) are the two most widely adopted scar-
less assembly methods. Both methods require that the plasmid
backbone into which the DNA fragments are to be assembled
be linearized and all DNA fragments to be assembled are linear
and overlap each other by 2060 base pairs. Overlapping frag-
ments are created by polymerase chain reaction (PCR) ampli-
cation of each fragment to be assembled using custom
oligonucleotide primers with 50 extensions that introduce iden-
tity to fragment that is required to be adjacent after assembly
(Figure 4). For Gibson Assembly all linear fragments are
combined in a single reaction. The reaction mix uses a cocktail
of three enzymes: T5 exonuclease performs a 50 to 30 resection
of DNA fragments creating 30 overhangs. These 30 overhangs are
able to self-anneal in the regions of overlap. A proofreading
DNA polymerase is included to extend the 50 ends, lling in
the gaps, and the nal enzyme, a thermostable DNA ligase
from Thermus aquaticus (Taq ligase), repairs the remaining
single-stranded nicks (Figure 4) resulting in a double-
stranded circular molecule that is transformed into E. coli for
replication. For assembly by SLIC all fragments are separately
treated with a single enzyme, T4 DNA polymerase, which, in
the absence of deoxynucleoside triphosphates (dNTPs),
exhibits 30 to 50 exonuclease activity. The addition of a single
nucleoside triphosphate, typically deoxycytidine triphosphate
(dCTP), is added to halt the resection, reverting T4 to a DNA
polymerase. In the absence of the full complement of dNTPs,
the enzyme will stall leaving 50 overhangs on all fragments.
The resectioned fragments are then combined together allow-
ing the overlaps to self-anneal. The reaction is transformed

Figure 3 Several series of plasmids (including binary plasmid vectors with origins of replication for Escherichia coli (ori Ec) and Agrobacterium
tumefaciens (ori At) as well as Left and Right Border (LB and RB) sequences) that enable a single coding sequence to be cloned in a single step are
still in common use. These typically contain a plant selectable marker gene close to the LB and a promoter and terminator interrupted by (a) a multi-
ple cloning site or (b) a Gateway cloning cassette consisting of a negative selection gene (ccdB) to prevent cells that have not acquired the DNA frag-
ment of interest between their site-specic recombination sites (att sites) in a clonase reaction with the ENTRY plasmid containing the DNA sequence
to be cloned.
116 Biotechnology j Synthetic Biology and Gene Cloning

Multiple linear fragments

with >20 bp overlaps

Sequence and Ligation Independent Cloning Gibson assembly

Linear fragments are separately treated with T4 DNA polymerase. All components are combined with a three enzyme cocktail:
In the absence of dNTPs, T4 induces a 3' to 5' resection. 1) T5 exonuclease performs a 5'-3' resection of all components
dCTP is added to arrest the reaction. allowing complementary overhangs to self-anneal.

The resectioned fragments are 2) A proof-reading DNA polymerase extends the resection
mixed together resulting in a plasmid with 3) Taq ligase fills in and join gaps
single-stranded nicks, which are repaired in E. coli.

Figure 4 Overlapping linear fragments of DNA, including a plasmid backbone, are created by polymerase chain reaction (PCR) amplication using
oligonucleotide primers with 50 extensions with identity to the adjacent fragment. For assembly by Sequence and Ligation Independent Cloning
(SLIC) overlapping, linear fragments and a plasmid vector backbone are separately treated with T4 polymerase. In the absence of dNTPs this
produces 50 overhangs. The exonuclease activity is halted by the addition of dCTP allowing the fragments to self-anneal into a seamless assembly.
For Gibson assembly, sequences are combined in a three-enzyme cocktail that rst creates 30 overhangs, allowing the products to self-anneal before
joining them in a seamless assembly. Redrawn and adapted from Patron, N.J., 2014. DNA assembly for plant biology: techniques and tools. Curr.
Opin. Plant Biol. 19C, 1419, published by Elsevier.

Figure 5 (a) Two BioBricks are digested with pairs of enzymes to produce compatible ends. The ligation product is a new BioBrick part consisting
of the original parts separated by a six base pair scar. (b) Multiple sequences are assembled in a single step by Golden Gate cloning into a backbone
containing pairs of inverted Type IIS restriction endonuclease recognition sequences. The recovered vector can be assembled with further fragments
in subsequent Golden Gate cloning reactions.

into E. coli where the nicks are repaired during plasmid replica-
tion (Figure 4).
The rst widely adopted biological assembly standard was
the BioBrick standard 10, implemented in the iGEM competi-
tion, an international synthetic biology competition in which
teams of students complete bioengineering projects using parts
stored in the Registry of Standard Biological Parts house at the
Massachusetts Institute of Technology (MIT). To date, tens of
thousands of BioBricks have been deposited at the Registry,
and variations and improvements on the original BioBrick

standard have been developed. In all BioBrick assembly
methods, individual DNA sequences are standardized by the
removal of recognition sequences for the restriction enzymes
used in the assembly protocol. Prex and sufx sequences are
then added to the start and end of the sequence. In the widely
used BioBrick standard 10, the prex contains EcoRI and XbaI
recognition sites and the sufx contains SpeI and PstI sites.
SpeI produces an overhang complementary to XbaI allowing
two BioBricks to be assembled together (Figure 5). In each
assembly reaction, one part is digested with the enzymes in
the prex allowing another part, released from its backbone
with the enzyme producing the complementary overhangs, to
be assembled upstream of it. The result is a new BioBrick part
containing both original parts separated by a small scar
(Figure 5). In contrast, Type IIS-mediated assembly methods
enable parallel assembly of multiple DNA parts in a one-pot,
one-step reaction. Unlike Type II restriction enzymes Type IIS
restriction enzymes, such a BsaI, cleave up- or downstream of
their non-palindromic recognition sequence. As the overhangs
produced by digestion do not contain any part of the recogni-
tion sequence, the overhang may comprise any sequence. The
production of multiple overhangs on different fragments with
the same enzyme allows multiple parts to be assembled in
a predetermined order and orientation in the same reaction
using only one restriction enzyme (Figure 5). The one-step
digestionligation reaction can be performed with any collec-
tion of plasmid vectors and parts, provided the DNA fragments
are housed in plasmids anked by a convergent pair of Type IIS
recognition sequences. Any plasmid vector can be converted for
this type of assembly method by the removal of any instances
of the Type IIS enzymes used for cloning other than a divergent
Biotechnology j Synthetic Biology and Gene Cloning 117
pair of recognition sequences for the same enzyme between
which the part or parts will be assembled. The parts must
also be free of recognition sites for this restriction enzyme
and be housed in a plasmid backbone with a different antibi-
otic resistance to the accepting plasmid into which parts will
be assembled.
See also: Plant Breeding and Genetics: Gene Editing
Technologies ZFNs, TALENs, and CRISPR/Cas9;
Transformation and Transgene Expression; Transformation in
Plastids.
Further Reading
Ellis, T., Adie, T., Baldwin, G.S., 2011. DNA assembly for synthetic biology: from parts
to pathways and beyond. Integr. Biol. 3, 109118.
Fesenko, E., Edwards, R., 2014. Plant synthetic biology: a new platform for industrial
biotechnology. J. Exp. Bot. 65, 19271937.
Gelvin, S.B., 2003. Agrobacterium-mediated plant transformation: the biology behind
the gene-jockeying tool. Microbiol. Mol. Biol. Rev. 67, 1637.
Lee, L.-Y., Gelvin, S.B., 2008. T-DNA binary vectors and systems. Plant Physiol. 146,
325332.
Patron, N.J., 2014. DNA assembly for plant biology: techniques and tools. Curr. Opin.
Plant Biol. 19C, 1419.
Verma, D., Daniell, H., 2007. Chloroplast vector systems for biotechnology applica-
tions. Plant Physiol. 4, 11291143.
Relevant Website
http://parts.igem.org The Registry of Standard Biological Parts.