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Binary Vectors and Super-binary Vectors Binary Vectors and Super-binary Vectors
Toshihiko Komari, Yoshimitsu Takakura, Jun Ueki,
Norio Kato, Yuji Ishida, and Yukoh Hiei
A binary vector is a standard tool in the transformation of higher plants mediated by Agrobacterium tumefaciens. It is composed of the borders of T-DNA, multiple cloningsites, replication functions for Escherichia coli and A. tumefaciens, selectable markergenes, reporter genes, and other accessory elements that can improve the efficiency ofand/or give further capability to the system. A super-binary vector carries additionalvirulence genes from a Ti plasmid, and exhibits very high frequency of transformation,which is valuable for recalcitrant plants such as cereals. A number of useful vectors arewidely circulated. Whereas vectors with compatible selectable markers and convenientcloning sites are usually the top criteria when inserting gene fragments shorter than 15 kb,the capability of maintaining a large DNA piece is more important for considerationwhen introducing DNA fragments larger than 15 kb. Because no vector is perfect forevery project, it is recommended that modification or construction of vectors should bemade according to the objective of the experiments. Existing vectors serve as goodsources of components.
Key Words: Agrobacterium tumefaciens; transformation; binary vector; super-binary
Research projects that involve transformation of higher plants are lengthy, complicated processes, which may last for years. The first parts of the projectsare the steps of vector construction, performed by molecular biologists. Com-pared with the entire durations of the projects, these steps are relatively shortand usually can be completed within weeks. However, scientists could make aseries of irrevocable decisions and sometimes mistakes in these fate-deter-mining steps, and the consequences would not emerge until years later.
From: Methods in Molecular Biology, vol. 343: Agrobacterium Protocols, 2/e, volume 1 Edited by: Kan Wang Humana Press Inc., Totowa, NJ 4/20/06, 10:53 AM Komari et al.
Researchers often find undesired aspects of vector configuration when charac-terizing progeny of transgenic plants; in a worst case scenario these vectordefects could ruin the entire project. In this chapter, steps from designing ofvectors through preparation of strains of Agrobacterium tumefaciens ready forinfection are addressed. It should be emphasized that good management ofthese processes can save a lot of precious time and resources.
Basic frameworks of the current vectors for transformation of higher plants were developed in the early and mid-1980s, soon after it had been elucidated
that crown gall tumorigenesis represented the genetic transformation of plant
cells (1). The first achievement was the removal of wild-type T-DNA, which
causes tumors and inhibits plant regeneration, from Ti plasmids to generate
"disarmed strains" such as LBA4404 (2). Earlier attempts at the introduction
of engineered T-DNA into A. tumefaciens involved the placement of genes in
E. coli vectors that could be integrated into a disarmed Ti plasmid (1). This
was a reasonably efficient system, but a limitation was that the final product is
a plasmid larger than 150 kb in A. tumefaciens, and confirmation of the struc-
ture was not straightforward.
Then the binary vector system was invented, exploiting the fact that the process for transfer of T-DNA is active even if the virulence genes and the
T-DNA are located on separate replicons in an A. tumefaciens cell (2).
An artificial T-DNA is constructed within a plasmid that can be replicated in
both A. tumefaciens and E. coli. Plasmid construction is completed in E. coli,
and simple transfer of the vector to A. tumefaciens produces a strain ready for
plant transformation. Soon such binary vectors were widely distributed among
plant scientists. Although the term binary vector literally refers to the entire
system that consists of two replicons, one for the T-DNA and the other for the
virulence genes, the plasmid that carries the T-DNA is frequently called a
binary vector. We follow this popular and convenient terminology in this
chapter.
One of the approaches toward enhancing the frequency of transformation by binary vectors is to employ additional virulence genes, such as virB, virE, and
virG, which exhibit certain gene dosage effects (3–6). In the super-binary vec-
tor system, a DNA fragment that contains virB, virC, and virG from pTiBo542
is introduced into a small T-DNA-carrying plasmid (7). A. tumefaciens strains
that carry pTiBo542 are wider in host range and higher in transformation effi-
ciency than strains that carry other Ti plasmids, such as pTiA6 and pTiT37 (8).
Super-binary vectors are highly efficient in the transformation of various plants
(see Note 1) and played an important role when the host range of transforma-
tion mediated by A. tumefaciens was extended to important cereals in the mid-
1990s (9,10). The final step of construction of a super-binary vector is
integration of an intermediate vector with an acceptor vector in A. tumefaciens,
4/20/06, 10:53 AM Binary Vectors and Super-binary Vectors but, unlike the aforementioned integration system, the final product in thesuper-binary vector system is a plasmid that can be confirmed by routinerestriction analysis of mini-scale DNA preparation from A. tumefaciens.
Commonly used binary and super-binary vectors are listed in Table 1. Help-
ful guidance for selection of the vectors has already been provided in the litera-
ture (11). Still, which is the best vector is a question with no definitive answer.
Since these are vehicles for delivery of transgenes to plants, they should be
(1) easy for the researcher to insert genes (loading), (2) efficient in plant trans-
formation (unloading), (3) widely available to researchers, and (4) versatile for
diverse purposes. Because such vectors can also be source materials for new
vectors, if any vector components can be easily replaced or removed, the vec-
tor will be very useful. Recently constructed vectors provide a number of user-
friendly features related to transgene loading and unloading, such as wide
selection of cloning sites, high copy numbers in E. coli, high cloning capacity,
improved compatibility with strains of choice, wide pool of selectable markers
for plants, and high frequency of plant transformation. However, our quick
survey of some 130 recently published papers, in which transformation of
higher plants mediated by A. tumefaciens was described, revealed that deriva-
tives of a relatively old vector, pBin19 (12), such as pBI121 (13), pIG121Hm
(9), and others, were still used in about 40% of these studies. One reason could
be that these vectors were widely circulated at early stages of plant transforma-
tion, and accumulated data in the literature from their use has built a lot of
confidence. Another reason might be a convenient feature of pBI121, namely,
that one-step replacement of the β-glucuronidase (Gus) gene with another gene
can quickly create an overexpression vector for the gene.
No matter how difficult the choice is, we have to make a decision. In our laboratories, the key criteria for delivery of transgene fragments smaller than
15 kb are (1) compatibility of selectable markers with the experiments and
(2) availability of convenient cloning sites. For delivering DNA fragments
larger than 15 kb, the top consideration is whether the large DNA fragments
can be cloned efficiently to the vectors and maintained stably in E. coli and
A. tumefaciens, because large DNA pieces in certain vectors, e.g., high-copy-
number vectors, may sometimes cause low efficiency of transformation of bac-
teria or rearrangement of the inserts (14).
The scope of transformation experiments in higher plants is complex, cover- ing topics such as overexpression, regulated expression, downregulation orshut-down of foreign or internal genes, expression of gene fusion, assays ofpromoters or other regulatory elements, complementation of mutations withgenomic sequences, tests of new molecular tools, tests of novel tissue cultureprotocols, and so on, with ever growing complexity. Therefore, in regard tothe versatility of the vector, it is futile to try to design a vector that can be 4/20/06, 10:54 AM Komari et al.
BIBACHomePage.html GenBank accession number, Contact AF485783, www.cambia.org A. tumefaciens pTiT37 (synthetic) Indication of more than one marker genes for a series of vectors means availability of a vector with each one of the markers.
Commonly Used Binary and Super-binary Vectors
4/20/06, 10:54 AM Binary Vectors and Super-binary Vectors suitable for all purposes. It is a good idea to modify existing vectors or to buildnew ones from scratch for specific purposes as demands arise.
2. Materials
2.1. Components of a Binary Vector
The compositions of widely circulated binary vectors are similar, and many of the components have been used for more than 15 yr without much modifica-
tion in plant transformation experiments. Therefore, the components listed in
this section are considered quite reliable. It is useful to prepare series of unfin-
ished plasmids that carry various combinations of these elements as materials
for molecular construction beforehand (see Note 2).
2.1.1. On the T-DNA 1. T-DNA borders and their sequence contexts. These components are usually DNA fragments cloned from well-known Ti plasmids. Imperfect, direct repeats of
25 bases, the right border (RB) and left border (LB), are said to be the only
essential cis elements for T-DNA transfer (15) but factors that enhance (over-
drive) or attenuate T-DNA transfer have been identified near the RB (16,17) or
the LB (17), respectively. Therefore, it may be safe to retain a few hundred bases
of natural sequences adjacent to the T-DNA. Sources for the borders are also
indicated in Table 1. Data for the border sequences are available in GenBank,
accession numbers ATU237588 for pTiC58, and ATACH5 for pTi15955 (see Note 3).
2. Multiple cloning sites (MCS). Many of the vectors have the MCS derived from popular cloning vectors, such as pUC8/9, pUC18/19, and pBluescript, whereasothers have unique sequences. The MCS from these standard vectors are conve-nient because gene components are usually cloned in such vectors beforehand.
Some vectors have the lacZ unit from the standard vectors, and blue/white selec-tion for insertion of fragments may be employed.
3. Selectable marker gene cassette for plant transformation (see Note 4).
a. Promoters. Selectable markers need to be expressed in calli, in cells from those plants that are being regenerated, or germinating embryos to facilitate
plant transformation. Therefore, promoters for constitutive expression are pre-
ferred. Promoters used mainly for dicotyledonous plants include the 35S pro-
moter from cauliflower mosaic virus (18) and promoters derived from Ti
plasmids, such as nopaline synthase (Nos) (19), octopine synthase (Ocs),
mannopine synthase (Mas), gene 1, gene 2, and gene 7 (20). Popular promot-
ers for monocotyledonous plants include the 35S promoter and the promoters
from the ubiquitin (Ubi) gene of maize (21) and the actin (Act) gene of rice
(22). The choice of promoters that drive the selectable marker genes affects
the efficiency of transformation. For example, the Ubi promoter gave a fre-
quency of transformation much higher than that of the 35S promoter in cere-
als (23; unpublished results).
b. Selectable markers for plants. Marker genes used in binary and super-binary vectors are listed in Table 2. Depending on the plants to be transformed,
4/20/06, 10:54 AM Komari et al.
Selection pressure bispyribac-sodium Kanamycin, G418, paromomycin Mannose as sole carbon source Bleomycin, phleomycin Glyphosate (Round-up) Neomycin phosphotransferase II Aminoglycoside 3' phosphotransferase II Aminoglycoside phosphotransferase IV Aminoglycoside phosphotransferase I Aminoglycoside phosphotransferase III Phosphinothricin acetyl transferase Phosphomannose isomerase Bleomycin binding protein Mutant dihydropteroate synthase Blasticidin deaminase Mutant acetolactate synthase Mutant acetohydroxy acid synthase Dihydroforate reductase Isopentenyl transferase Aminoglycoside nucleotidyl transferase β-Lactamase Tetracycline efflux protein Cyanamide hydratase Selectable Markers (S) and Reporter Genes (R) Employed in Binary and Super-binary Vectors
Common abbreviations NptII, Aph 3' II, Kan Hpt, Hph, AphIV, Hyg 4/20/06, 10:54 AM Binary Vectors and Super-binary Vectors viewed in the literature 4-Methyl tryptophan D-Xylose as sole carbon source Butafenacil (herbicide) Tryptophan decarboxylase Mutant glutamete-1-semialdehyde aminotransferase 3,5-Dibromo-4-hydroxybenzoic acid nitrilase Mutant protoporphyrinogen oxidase β-Glucuronidase Luciferase Green fluorescent protein β-Galactosidase Nopaline synthase Anthocyanin Oxalate oxidase A number of useful, distinctive derivatives of GFP and fluorescent proteins with different characteristics are available and rea 4/20/06, 10:54 AM Komari et al.
the choice of selectable markers greatly affects the efficiency of transforma-
tion, and restrictive/permissive concentrations of selective agents vary con-
siderably among plant species and even among cultivars (see Note 5).
Kanamycin resistance is good for many dicotyledons including tobacco,
tomato, potato, and Arabidopsis (see Note 6). Hygromycin resistance (hpt) is
very good for rice transformation (9), and the phosphinothricin resistance gene
(bar) is efficient for maize and other cereals (10,24). We do not think the
effects of selectable markers have been explored sufficiently, because many
of them were tested in very limited species of plants. Therefore, we made a
comprehensive list of marker genes in Table 2, hoping that some of the mark-
ers may be investigated further to improve transformation of certain plant
species.
c. 3' Signal (see Note 7): DNA fragments of a few hundred bases derived from
the 3' ends of the CaMV 35S transcript and Agrobacterium Nos and otherT-DNA genes are carried by many of the binary and super-binary vectors.
2.1.2. On the Vector Backbone 1. Bacterial selectable marker gene. Genes that can confer resistance to kanamycin, gentamycin, tetracycline, chloramphenicol, spectinomycin, and hygromycin are
popular markers for bacterial selection for both E. coli and A. tumefaciens (Table 2).
Care must be exercised as some bacterial strains without vector plasmids have
certain intrinsic antibiotic resistance. Some selectable markers for plants, such as
Nos-nptII and 35S-hpt, give fair levels of resistance to both E. coli and A. tumefaciens
(see Note 8). If such a dual function gene is present in the T-DNA, bacterial
selectable markers may be omitted from the vector backbone to simplify the vec-
tor construction.
2. Plasmid replication functions. Binary vectors need to be replicated both in E. coli and A. tumefaciens. Use of plasmid replication functions with a wide host range,
such as the ones of plasmid incompatibility group P (IncP) or W (IncW) is a good
option. IncP binary vectors carry the origin of vegetative replication (OriV) and
the transacting replication functions (Trf) of IncP plasmids (25). The replication
locus of IncW plasmids, such as pSa, consists of the origin of replication and
RepA gene (26). pGreen vectors have only the origin of replication, and the RepA
function is provided by another plasmid, pSoup, in A. tumefaciens (27). Alterna-
tively, replication functions for A. tumefaciens, such as the ones for an Ri plas-
mid (28) or pVS1 (29), and for E. coli, such as the ones for the F factor, phage P1,
ColE1, or P15A (14), may be combined. Types of replication functions deter-
mine copy numbers and stability of the plasmids in bacterial cells. The use of
high-copy plasmids for cloning of fragments larger than 15 kb can sometimes
result in complications like rearrangement of DNA. If this is a problem, a good
choice is an IncP plasmid, such as pBI121, which is a low-copy-number (about
five copies per cell) plasmid in both E. coli and A. tumefaciens (see Note 9).
3. Plasmid mobilization functions (see Note 10). The origin of transfer (OriT) of
IncP plasmids (25) or the bom function of ColE1 plasmid (14) is carried by most
4/20/06, 10:54 AM Binary Vectors and Super-binary Vectors of the binary vectors. Plasmids with one of the sequences may be mobilized from
E. coli to A. tumefaciens aided by a conjugal helper plasmid, pRK2013 or
pRK2073 (30). pRK2073 is a derivative of pRK2013 and has an insertion of Tn7
in the kanamycin resistance gene of pRK2013. Either one works fine for most
applications, but pRK2073 is recommended if the target plasmid carries kanamy-
cin resistance, and pRK2013 is recommended if the target has spectinomycin
resistance.
2.2. Components of a Super-binary Vector
2.2.1. Intermediate Vector 1. T-DNA. The same composition described for a binary vector applies (see Sub-
2. Plasmid replication. An intermediate vector has an origin of replication of ColE1 like pBR322 and is replicated in E. coli but not in A. tumefaciens.
3. Plasmid mobilization. An intermediate vector has the bom function of ColE1 near the replication origin and can be mobilized by pRK2013 or pRK2073.
4. Bacterial selection. An intermediate vector has a spectinomycin resistance gene derived from Tn7 (31).
5. Homology with an acceptor vector. An intermediate vector and an acceptor vec- tor share the 2.7-kb fragment that contains the ori and bom of ColE1 and the cos
site from phage lambda (14) (the Ori-Cos fragment).
2.2.2. Acceptor Vector 1. Plasmid replication. An acceptor vector is an IncP plasmid and also has the ori of 2. Plasmid mobilization. An acceptor vector has both the bom function of ColE1 and OriT of IncP plasmids and can be mobilized aided by pRK2013 or pRK2073.
3. Bacterial selection. An acceptor vector has tetracycline resistance derived from 4. Virulence genes. An acceptor vector has a 14.8-kb KpnI fragment (the super-vir fragment) from pTiBo542. This fragment contains virB, virC, and virG operons
(see Note 1).
5. Homology with intermediate vectors (see Subheading 2.2.1.).
6. T-DNA with a plant-selectable marker gene. An acceptor vector optionally has a
T-DNA to create a vector for cotransformation.
2.3. Reporter Gene Cassette
1. Promoters. It is convenient to have a reporter gene expressed in various tissues and organs, and so the promoter is often chosen from the same group of promot-ers that may be used for selectable marker genes, which include 35S, Ubi, Act,Nos, and other T-DNA promoters. In some of the vectors, the promoter for theselectable marker and the reporter is the same, but, generally speaking, avoid-ance of duplication of the same components is recommended.
4/20/06, 10:54 AM Komari et al.
2. Reporter genes. β-Glucuronidase (Gus) (13), green fluorescent protein (GFP) (32)
and luciferase (Luc) (33) are the most popular reporter genes (Table 2). Back-
ground activities in the assays of these enzymes are generally very low in higher
plants. Reporter genes can be linked to regulatory sequences and used to study
functionality of these sequences. Because Gus and GFP are highly stable proteins
in plant cells (34), the activity of these proteins may not immediately reflect small
or quick changes in the level of the mRNA for these proteins in plant cells. If this
is the case, Luc, whose half-life in plant cells is much shorter than those of Gus
and GFP (34), may be a good choice. A reporter gene that has an intron in the
coding sequence, such as the intron-Gus gene (35), is very useful because this
gene is not expressed in A. tumefaciens. In addition, especially in monocotyle-
dons, introns can enhance expression for some genes (36). Introns placed close to
the N-terminal in the coding sequence and in the 5' untranslated region of a gene
may be equally effective (37).
3. 3' Signal. The 3' signal for a reporter gene may be chosen from the components listed for the selectable marker genes (see Subheading 2.1.1.).
2.4. Accessory Components for Binary and Super-binary Vectors
1. Restriction sites for endonucleases with long recognition sequences. Because genes have various restriction sites, it may not always be easy to find unique sites
for introducing DNA sequences to a desired location on a vector. More than 10
restriction enzymes that recognize 8 bases are available now, and there are sev-
eral homing endonucleases, which have recognition sites longer than 10 bases.
It is useful to design vectors with a number of sites for these enzymes. Such
vectors are especially useful for stacking of multiple expression units in one vec-
tor. Well-designed sets of plasmids that consists of a binary vector with these
sites and high-copy cloning vectors with expression cassettes and subsets of the
sites are called as modular vectors (38).
2. The sites for the GATEWAY® system. Molecular cloning based on restriction enzymes and DNA ligases is not always straightforward. The GATEWAY sys-
tem (Invitrogen) is a cloning technology based on the site-specific recombination
system of phage lambda. A step of molecular cloning may be performed in a
single tube within a few hours, and E. coli that carries a desired plasmid is recov-
ered at a very high frequency on the following day. In essence, a DNA fragment
flanked by a pair of short, specific sequences may easily be replaced with another
DNA fragment by the GATEWAY system. By placing the GATEWAY recombi-
nation sites at appropriate locations in the vectors, workload for subsequent clon-
ing steps may be greatly reduced. Convenient binary vectors based on the
GATEWAY technology have been reported by various authors (39,40).
3. Virulence genes. Small DNA fragments that contain virE or virG can improve the efficiency of transformation by a binary vector to some extent (4,5). A mutant
virG gene, virGN54D, that is expressed constitutively in Agrobacterium cells
gave much higher efficiency of transformation than wild types (41,42).
4/20/06, 10:54 AM Binary Vectors and Super-binary Vectors 4. Device to suppress transfer of non-T-DNA segments.
a. Multiple left border repeats. Transfer of so-called "backbone sequences" from binary vectors to higher plants is not uncommon and has raised considerable
concerns over genetically modified plants. A simple method is to place addi-
tional LB sequences close to the original LB; transfer of the backbone
sequences is then suppressed in a nearly perfect fashion (43).
b. Killer gene. Another method is to place a gene, whose gene product is lethal to cells, outside the T-DNA to eliminate transformed cells that acquired the
backbone (44).
5. Cosmid. The cloning capacity for a cosmid is up to about 50 kb, which includes the vector DNA, being based on the packaging system of phage lambda (14). A simple
addition of one or two copies of cos sites can convert a binary vector to a cosmid.
6. P1 Cloning system. A P1 vector is more complex than a cosmid, and several components need to be integrated. However, P1 vector is very useful in genomic
studies because the cloning capacity for a P1 vector is as large as 100 kb, being
based on the packaging system of phage P1 (14).
7. Device for removal of selectable marker genes.
a. Cotransformation. Considerable concern has been raised over selectable marker genes in commercial transgenic plants. Cotransformation with two
separate T-DNAs is a simple approach for removal of the marker gene.
One T-DNA carries a selectable marker gene, and the other does genes of
interest. There is a good chance that these T-DNAs, segregate independently,
and marker-free progeny plants are identified. Two T-DNAs may easily be
constructed on a super-binary vector (45). The only modification is that a
T-DNA with a selectable marker gene is cloned into a precursor of an accep-
tor vector before the virulence fragment is inserted and an intermediate vector
is prepared without a selectable marker gene.
b. Site-specific recombination systems. Many authors have reported vectors exploiting site-specific recombination systems derived from phages or fungi,
such as the Cre-lox, Flp-FRT, and R/RS (46). In such a vector, a marker gene
is flanked by the short target DNA sequences for a specific recombinase. After
the integration of the T-DNA to plant cells, the recombinase is provided to the
cells by various sophisticated means so that the marker gene is excised out.
8. Accommodation for very large DNA segments. For map-based cloning of plant genes, complementation tests of large genomic fragments provide key informa-
tion. Single-copy vectors that carry Ri ori for A. tumefaciens and F ori or P1 ori
for E. coli were specifically designed for transfer of very large DNA fragments to
higher plants and designated as BIBAC (47) and TAC (48). On the other hand,
because a simple IncP binary vector was able to maintain DNA fragments stably
over 300 kb (49), conventional binary vectors that do not carry the ori of ColE1
or pUC may also be good for this purpose. We think the current situation is that
cloning and transfer of DNA fragments larger than 50 kb is possible, but the
efficiency is still low with any of these vectors.
4/20/06, 10:54 AM Komari et al.
3. Methods
3.1. Construction of a Typical Binary Vector
A standard flowchart showing the construction of binary vectors, from vari- ous components to the creation of an empty vector, a vector with a plant-select-
able marker (selection vector), and finally a vector with both a reporter gene
and a selectable marker (reporter vector) is illustrated in Fig. 1. The reporter
gene in this flowchart may be a model for any genes of interest. In transforma-
tion experiments, a reporter vector may serve as a control vector, which gives
reference points for virtually all important measurements in transformation
processes, such as frequency of transformation, growth of transformed cells,
efficiency of plant regeneration, growth of transgenic plants, phenotypes of
plants, level of foreign gene expression, effects of genes of interest, and so on
(see Note 11). Our recommendation is to start the consideration of experimen-
tal designs from the configuration of such a control vector. Quite often, a reporter
vector may be a good starting material for various gene constructs, as experi-
mental vectors may be obtained simply by replacing one or more components
in the reporter vector with appropriate DNA fragments. Useful tips related to
vector construction are given in Notes 12–18.
1. Obtain plasmids and other DNA fragments necessary for constructions of vectors from appropriate sources.
2. Combine the bacteria-selectable marker and the plasmid replication functions for E. coli.
3. Insert the plasmid replication functions for A. tumefaciens, if necessary.
4. Insert the plasmid mobilization functions, if necessary.
5. Insert the RB, the LB, and the MCS to give the empty vector.
6. Construct the expression unit of the selectable marker gene separately.
7. Insert the unit into the empty vector to give the selection vector.
8. Construct the expression unit of the reporter gene separately.
9. Insert the unit into the selection vector to give the reporter vector.
3.2. Examples of Binary Vectors
Some examples of binary vectors are shown in Fig. 2. Various derivatives
differing in the selectable marker, reporter, MCS, and other factors are avail-
able in the pCAMBIA series (www.cambia.org), pGreen series (27), and pPZP
series (50). One of the derivatives is shown for each of the groups; empty vector
pGreen0000, selection vector pPZP111, and reporter vector pCAMBIA1302.
A similar variation is found in the derivatives of pBin19, which is considered
to be a selection vector, and reporter vector pBI121, shown in Fig. 2. An empty
version of pBin19 may be obtained by digesting pBin19 with ClaI and partially
with SacII, followed by recircularization. These vector groups have been
4/20/06, 10:54 AM Binary Vectors and Super-binary Vectors Fig. 1. A simplified flowchart showing the construction of binary vectors.
successfully employed in many studies, which, at a glance, account for two-thirds of the recent publications in the area of plant transformation.
3.3. Construction of a Typical Super-binary Vector and Examples
The system of a super-binary vector consists of two plasmids, an intermedi- ate vector and an acceptor vector, and the final construct is a cointegrate plas-
mid created by homologous recombination in A. tumefaciens (45). Protocols
for both a single T-DNA vector, in which a selectable marker and a gene of
4/20/06, 10:54 AM Komari et al.
Fig. 2. Examples of binary vectors. The maps are based on sequences in GenBank, accessions numbers CVE7829 for pGreen0000, CVU10487 for pPZP111, AF485783for pBI121, and AF234298 for pCAMBIA1302. Abbreviations: RB, right border;LB, left border; P35S, promoter for 35S transcript; 3' 35S, 3' signal for 35S transcript;PNos, promoter for nopaline synthase; 3' Nos, 3' signal for nopaline synthase; nptII,neomycin phosphotransferase II; Gus, β-glucuronidase; hpt, hygromycin phospho-transferase; lacZ, α-subunit of β-galactosidase; IncW, origin of replication of IncWplasmid; pVS, origin of replication of pVS1; OriV, origin of vegetative replication ofIncP plasmid; Trf, transacting replication function of IncP plasmid; OriT, origin oftransfer of IncP plasmids; ColE1, origin of replication of ColE1; Bom, bom site forplasmid transfer of ColE1; CmR, chloramphenicol resistance gene; KanR, kanamycinresistance gene.
4/20/06, 10:54 AM Binary Vectors and Super-binary Vectors interest are linked in a T-DNA, and a double T-DNA vector for cotransforma-tion are described here.
3.3.1. Single T-DNA Vector 1. Construct an empty intermediate vector by combining the Ori-Cos fragment, the spectinomycin resistance gene from Tn7, the cos site of phage lambda, the RB
and the LB from pTiT37, and the MCS. pSB11 (Fig. 3) is an example.
2. Clone a gene of interest and a plant-selectable marker gene into the MCS of the empty vector.
3. As a preliminary step in the construction of an acceptor vector, combine the Ori- Cos fragment, the tetracycline resistance locus, OriV, Trf, OriT, and an MCS that
consists of XbaI, SacI, XhoI, KpnI, and HindIII recognition sites to give a precur-
sor plasmid. pNB1 is an example (Fig. 3).
4. Clone the Super-vir fragment into the KpnI site of the precursor plasmid to give an acceptor vector (see Note 19). pSB1 (Fig. 3) is an example.
5. Introduce the derivative of the intermediate vector into an A. tumefaciens strain that carries the acceptor vector by triparental mating, and select a strain for
spectinomycin and tetracycline resistance and growth on a minimal medium to
create the cointegrate (see Note 20).
3.3.2. Double T-DNA Vector 1. Clone separately a gene of interest and a plant-selectable marker gene into an empty intermediate vector.
2. Cut out the T-DNA of the selectable marker from the intermediate vector as a SalI fragment and clone into the XhoI site of the precursor plasmid described in
Subheading 3.3.1. (see Note 21).
3. Clone the Super-vir fragment into the derivative of the precursor plasmid to cre- ate an acceptor vector with the plant-selectable marker gene. pSB4U (Fig. 2)
(43), which has the hygromycin resistance gene connected to the Ubi promoter
and the Nos 3' signal, is an example.
4. Introduce the intermediate vector with the gene of interest into an A. tumefaciens strain that carries the acceptor vector with the plant-selectable marker gene bytriparental mating and select a strain for spectinomycin and tetracycline resis-tance and growth on a minimal medium to create the cointegrate.
3.4. Preparation of A. tumefaciens
with Binary and Super-binary Vectors Ready for Infection
After completion of molecular construction in E. coli, vectors are transferred to A. tumefaciens by the procedures described in Chapter 3 of this volume.
For the construction of super-binary vectors, triparental mating is highly rec-
ommended (see Note 20).
Rearrangement of vectors may sometimes take place during the process of introduction of plasmids into A. tumefaciens. It is very important to confirm 4/20/06, 10:54 AM Komari et al.
4/20/06, 10:54 AM Binary Vectors and Super-binary Vectors the structure of vectors in colonies of A. tumefaciens and select the coloniesthat carry the right vectors. Once the right colonies are identified, the vectorsare reasonably stable in A. tumefaciens.
Ideally, plasmids are purified from A. tumefaciens and extensively character- ized by restriction analysis. However, preparation of plasmids from A. tumefaciensis much less efficient than that from E. coli, and a certain amount of experiencewith this protocol is needed to obtain good preparations of the plasmids. Alterna-tively, amplification of a number of key fragments of the vectors by polymerasechain reaction (PCR) from the colonies of A. tumefaciens or Southern analysis oftotal DNA preparation from A. tumefaciens cells is performed.
If something is wrong with the transformation vector, years of time, effort, and precious resources can be wasted. A laboratory should establish a series of
quality control (QC) protocols for vectors and bacterial strains (see Note 22).
Ideally, QC protocols are written down, and a member of the laboratory is
designated as the QC manager, who makes sure everyone follows the rules of
the laboratory.
1. The capability of a super-binary vector is most evident when it is combined with strain LBA4404, whereas the performance of a super-binary vector is not very
good when it is carried by strains derived from A281, such as EHA101, EHA105,
or AGL1 (6,9). The virC1 gene in the super-binary vector is probably inactive
owing to a frame-shift mutation that took place during the construction of vectors
(see GenBank accession number AB027255), but there is no evidence that this
affects the efficiency of transformation.
2. Useful examples of the plasmids are listed below, but do not try to make a com- plete set at one time. It is good enough to create plasmids as required and let thelibrary grow over time.
a. Minus-one vector: this vector lacks one of the components of the expression units of the vector.
b. Empty vector: the T-DNA of this vector has only MCS.
Fig. 3. (previous page) Examples of super-binary vectors and illustration of inte- gration of an intermediate vector to an acceptor vector. The maps of pSB11, pSB1,and pSB4U are based on GenBank accession numbers AB027256, AB027255, andAB201314, respectively. Abbreviations: RB, right border; LB, left border; U-hpt-N,Ubiquitin promoter-hygromycin phosphotransferase-3' signal for nopaline synthase;OriV, origin of vegetative replication of IncP plasmid; ColE1 or O, origin of replica-tion of ColE1; Trf, transacting replication function of IncP plasmid: OriT, origin oftransfer of IncP plasmid; Bom or B, bom site for plasmid transfer of ColE1; Cos or C,Cos site of phage lambda; TetR, tetracycline resistance gene; SpR, spectinomycinresistance gene.
4/20/06, 10:54 AM Komari et al.
c. High-copy MCS vector: a high-copy-number plasmid that has only MCS and no T-DNA borders. This may be convenient for creation of constructionintermediates, especially in case the vector to be constructed is a low-copy-number plasmid d. Marker cassette vector: the high-copy MCS vector that carries the expression unit of the plant-selectable marker gene.
e. Reporter cassette vector: the high-copy MCS vector that carries the expres- sion unit of the reporter gene.
3. The T-DNA border regions of nopaline-type plasmids, pTiC58 and pTiT37, are almost identical, and the T-DNA sequences of the well-studied octopine-type Ti
plasmids, pTi15955, pTiAch5, pTiA6, and pTiB6S3, are very similar to each
other if not identical (51,52).
4. In vectors constructed earlier, the selectable markers were located close to the RB. Because the transfer intermediate of the T-DNA is made in the direction
from the RB to the LB, it is considered that deletion of a gene of interest may be
prevented by placing a selectable marker close to the LB, and later most plasmids
were constructed in this way. In contrast, integration of T-DNA into a plant
chromosome is said to take place in the direction from the LB to the RB (53).
We have observed deletions at both the right and left ends of the T-DNA in
plants. Therefore, it is our opinion that the location of the selectable marker does
not matter much.
5. The use of weak promoters may not always be a bad idea. Strong promoters could waste resources for transcription and translation machinery in plant cellsafter transformation. In addition, because the levels of expression of markergenes and genes of interest are often linked, selection of transformants with weakselectable markers may cause strong expressers of the genes of interest to beobtained.
6. Expression of the nptII gene can inactivate a group of aminoglycoside antibiotics (14). Choice of antibiotic is an important factor in plant transformation. For example,
because kanamycin does not restrict growth of rice and maize cells, it is not used
for transformation of these plants. Many transformed rice cells resistant to G418
were albinos. However, rice and maize can be transformed reasonably well with
the nptII gene based on resistance to paromomycin (unpublished results).
7. The DNA segments connected to the 3' ends of genes are often called termina- tors, but the terminology is sometimes confusing, because signals for the termi-nation of transcription and for the addition of polyA sequences are different.
Exact functions of many 3' sequences are not well characterized and it is notusually confirmed whether termination signals are really contained in termina-tors. Therefore, we use the term 3' signal in this chapter.
8. Partly because the TATA boxes of eukaryotic promoters resemble prokaryotic promoters to some extent, many plant promoters are active in both E. coli and
A. tumefaciens (35). The fact that both the widely used strain EHA101 and the
vector pBin19 have kanamycin resistance had caused some inconvenience in ear-
lier days. One solution was insertion of the 35S-hpt gene to pBin19, which gave
4/20/06, 10:54 AM Binary Vectors and Super-binary Vectors another resistance to the vector and made introduction of the vector to the straineasier.
9. Subcloning of a gene into IncP plasmids can sometimes be a less efficient prac- tice compared with that of using the pUC derivatives. It is advisable to follow
faithfully the cloning procedures and tips described in standard textbooks such as
Molecular Cloning (14). Oversimplification or too many shortcuts during the
subcloning process may lead to complications and delay the completion of con-
struction. Sequence and other genetic information of IncP plasmids has been pre-
viously described (25). In general, IncP vectors that are larger than 20 kb can be
more stably maintained in both E. coli and A. tumefaciens than smaller IncP vec-
tors. We have observed that, after 3 d of culture without selective antibiotics,
most of the A. tumefaciens cells retained large IncP plasmids, whereas more than
half of the cells lost the small IncP vectors. How this phenomenon affects the
transformation experiments is not known. This can be important for Arabidopsis
in planta transformation, in which A. tumefaciens cells probably sit in plant tis-
sues for some time before the gene transfer process takes place.
10. Although plasmid mobilization functions are not needed for transformation of A. tumefaciens by electroporation or freeze-thaw methods, it is a good idea tohave broader options. When plasmid cointegration in A. tumefaciens is intended,triparental mating is much more efficient than electroporation, and these func-tions are necessary.
11. When plants are transformed with various gene constructs, it is a good idea to always transform plants in parallel with such a vector, which is extremely useful
in monitoring many aspects of transformation processes. Many factors are
involved in successful transformation, and it is not a simple task to maintain
capability of plant transformation stably over time. If something goes wrong in
experiments, what is happening in the control plots can answer many questions.
It is a good idea to include the same control vector in all the transformation
experiments conducted in a laboratory. We have been transforming rice for more
than 15 years now with A. tumefaciens. We can still compare data between cur-
rent and very early experiments if pIG121Hm (9) is included. Such a control
vector could also play the role of a "spearhead". New enabling technologies are
continuously developed, and new methods for plant transformation are tested
one after another. Each time, new factors are incorporated into mainstream pro-
tocols in a laboratory after they are tested extensively with the control vector.
12. Do not assume that external information related to biological materials is 100% correct. This is one of the hot spots for complications. You may receiveincorrect or incomplete maps, sequence information, protocols, and even plas-mids. The earlier you confirm materials and information after receipt, the moreeasily problems are identified and solved.
13. Characterize biological materials including plasmids and DNA fragments, and evaluate data collected in-house and external information as extensively aspractical. Ideally, everything from external sources is fully sequenced in-house.
At least, partial sequencing of the most critical segments and restriction analysis 4/20/06, 10:54 AM Komari et al.
with every six-base cutter enzyme available should be performed. The finishedvectors should also be characterized as described here.
14. Simulate vector construction in silico and prepare sequence files and maps of the vectors to be constructed before starting wet laboratory practices.
15. All fragments amplified by PCR must be fully sequenced.
16. It should be noted that similar genes, which consist of the same promoters, the same coding sequences, and the same 3' signals, could still be expressed quitedifferently even in the same plant species when they are placed in different vec-tors, probably being affected by small differences in the configurations of thevectors. Because the nature of these effects is not well understood, it is a goodidea to consider more than one molecular design and to use trial and error.
17. A prudent approach for experimental constructs is to make as few alterations as possible from a reporter vector, from which marker genes have been expressedvery well.
18. In the design of vectors, avoidance of repeats of sequences is highly recom- 19. The packaging extracts from phage lambda can greatly facilitate this cloning because the precursor is a cosmid and the size of the precursor plus the Super-virfragment is good for the packaging reaction.
20. Triparental mating (see Chapter 3) is a simple technique in principle but is some- times a hot spot of complications. The selection of A. tumefaciens from E. coli isoften based on capability of growth on a minimal medium. A certain backgroundgrowth of nontarget bacteria is inevitable on primary selection plates, but colo-nies that can grow as fast on the selective medium as on a nonselective mediumare clearly distinguishable. Second selection plates are usually clean, but it is agood idea to perform one more selection culture. After the third selection, plas-mids are prepared from the selected colonies as described in Chapter 3 in thisvolume, and restriction analysis is performed.
21. Although there are unique restriction sites in pSB1 and other acceptor vectors, direct cloning of additional DNA fragments into these vectors is not efficient. Therefore,if modification of an acceptor vector is necessary, it is a good idea to go back onestep to a precursor plasmid like pNB1, which lacks the Super-vir fragment. Aftermodification of the precursor plasmid, the 14.8 KpnI Super-vir fragment is clonedback. Again, the packaging extracts from phage lambda can facilitate this cloning.
It should also be noted that pUC plasmids carrying the Super-vir fragment aresometimes unstable, and pSB1 is a good source of this fragment.
22. The following points should be addressed in standard QC protocols: a. Source materials: it is a good idea to establish a central stock of a laboratory of commonly used plasmid DNA, bacteria, and other biological materials usedfor construction of vectors.
b. Constructed vectors and strains: DNA of finished vectors and all construction intermediates should be stored for a defined length of time. Purified plasmidDNA may be stored at –20°C for a very long time. Key plasmids are stored asDNA and E. coli with the plasmids.
4/20/06, 10:54 AM Binary Vectors and Super-binary Vectors c. Finished vector: a standard process for quality checking of vectors ready for plant transformation should be established. Thorough restriction analysis withmore than 10 enzymes and sequencing of junctions of fragments manipulatedduring the construction are minimum requirements. Important vectors shouldbe fully sequenced.
d. A. tumefaciens strain ready for transformation: the routine practice for confir- mation of structures of vectors in A. tumefaciens and the method of storage ofthe strains should be described.
e. Handling of A. tumefaciens: proper handling of bacterial strains recommended by bacteriologists may not always be exercised by current molecular biolo-gists. For E. coli, which outgrows virtually any organisms in laboratories, thisis usually not a problem, but for A. tumefaciens, practices like always usingfresh, well-isolated colonies, whose shapes and colors are carefully exam-ined, are important.
f. PCR primers and probes: quite a few pairs of primers and probe fragments are used during the construction. These are also useful for confirmation of vectorstructure and analysis of transgenic plants. It is a good idea to organize alibrary of these materials in a laboratory.
g. Bioinformatics: sequences, maps, and other data from various assays of the aforementioned materials constitute an enormous amount of electronic data,which should be well organized, regularly updated, and available to every-body in the laboratory.
We are grateful to Dr. Michael Bevan, Dr. Pal Maliga, Dr. Richard Jefferson, Dr. Csaba Koncz, Dr. Gynheung An, and Dr. Hongbin Zhang for agreeing tobe contacts for the vectors listed in Table 1. We thank Dr. Kan Wang for help-ful discussions and encouragement and Ms. Kumiko Donovan and Ms. AyakaYamashita for technical assistance.
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