DNA Cloning With Vector

What is meant by a Vector

Main features of vectors
Basic modes of actions
Main types of vectors
Plasmid used as vectors for Recombinant DNA technology
Types of plasmid vectors
Good plasmid cloning vehicles share a number of desirable features

IMPORTANT VECTORS USED FOR RECOMBINANT DNA TECHNOLOGY

pBR322 vector
Bacteriophage lambda

Two basic types of phage lambda vectors: insertional vectors and replacement vectors

DNA CLONING BY USING SINGLE STRANDED DNA VECTORS

Filamentous bacteriophages
Phage M13

VECTORS USED FOR CLONING LARGER FRAGMENTS OF DNA

Cosmids
BAC and PAC
Specialist type of vector
The Gateway® system is a highly efﬁcient method for transferring DNA fragments to a large number of different vectors

Conclusion

References

What is meant by a Vector ?

In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. … Of these, the most commonly used vectors are plasmids.

Main Features of Vector:

Origin of replication

For replication and maintenance of vector in host cells.

Promoter

To drive transcription of vectors transgene
Also to derive transcription of other genes in vector such as the antibiotic resistance genes.

Cloning sites

Allow for the insertion of foreign DNA into the vector through ligation.

Reporter genes

It allow for the identification of plasmid that contain inserted DNA sequences.

Targeting Sequences

That directs the expressed protein to the specific organelle in a cell or a specific location.

Protein purification tags

Some expression vectors contain proteins and peptide sequences that allow for easier purification of expressed protein.

Antibiotic resistance

Allow for the survival of cell that has been taken up by a vector in growth media containing antibiotics through antibiotic selection.

Epitope

Allows for antibody identification of cells expressing the target proteins,

Genetic markers

Allow for conformation that the vector has integrated with the host genomic DNA.

Basic Mode of Actions:

Vectors perform their functions in two ways mostly.

Transcription
Expression

Expression

There are two types of expression vectors

Prokaryotic expression vectors
Eukaryotic expression vectors

Prokaryotic expression vectors

Promoter
Ribosome binding sites
Translation initiation sites

Eukaryotic expression vectors

Polyadenylation tail
Minimum UTR length
Kozak sequence

Main Types of Vectors

In molecular biology a vector may refers to

Binary vector
Cloning vector
Shuttle vector
Expression vectors
Plasmid vector
Viral vectors

Binary Vectors

A transfer DNA (T-DNA) binary system is a pair of plasmids consisting of a binary plasmid and a helper plasmid. The binary vector is a shuttle vector, so-called because it is able to replicate in multiple hosts (E. coli and Agrobacterium tumefactions).

Cloning vector

A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloningpurposes. Cloning vectors in yeast include yeast artificial chromosomes (YACs).

Shuttle vector

A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species. Therefore, DNA inserted into ashuttle vector can be tested or manipulated in two different cell types.

Expression Vectors

An expression vector, otherwise known as an expressionconstruct, is usually a plasmid or virus designed for geneexpression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell’s mechanism for protein synthesis to produce the protein encoded by the gene.

Plasmid Vector

A genetic structure in a cell that can replicate independently of the chromosomes, typically a small circular DNA strand in the cytoplasm of a bacterium or protozoan. Plasmids are much used in the laboratory manipulation of genes.

Main Parts

Plasmids contain three components:

Origin of replication,
Polylinker to clone the gene of interest (called multiple cloning site where the restriction enzymes cleave),
An antibiotic resistance gene (selectable marker).

Plasmids used as vector in Recombinat DNA technology:

Following are the plasmids that are used for recombinant DNA technology.

Bacteriophages
- Bacteriophage λ derived vectors
- M13, f1, and fd filamentous phages derived vectors
Viral vectors for mammalian cells (especially used in gene therapy)
- Retroviruses
- Adeno-viruses
- Lentiviruses

Types of plasmid vectors:

Plasmids vectors may be:

Conjugative / transmissible vectors

They mediate DNA transfer through conjugation and therefore spread rapidly among the bacterial cells of a population e.g F plasmid,many R and some Col plasmid.

Non-conjugative vectors

They don’t mediate DNA through conjugation.

Ti Plasmid

A Ti or tumour inducing plasmid is a plasmid that often, but not always, is a part of the genetic equipment that Agrobacterium tumefaciens and Agrobacterium rhizogenes use to transduce its genetic material to plants.

Col Plasmid

Col plasmids, which contain genes that code for bacteriocins, proteins that can kill other bacteria.

Virulence Plasmid

Virulence plasmid turns the bacteria into a pathogen. So, they are responsible for carrying the genes which cause disease.

F Plasmid

The Fertility factor (first named F by one of its discoverers Esther Lederberg) allows genes to be transferred from one bacterium carrying the factor to another bacterium lacking the

factor by conjugation. The F factor is carried on the F episome, the first episome to be discovered.

R ( Resistance) plasmid

Resistance plasmid is a small element outside the chromosome that carries DNA information that fights against antibiotic drugs. An example of a resistance plasmid is pBR322 which carries the genes for tetracycline and ampicillin resistance.

Degradative plasmid

These plasmids are types of plasmid present in certain bacteria’s such as Pseudomonas putidawhich impart the ability to degrade Xenobiotic compounds.

For example

There are three such plasmids

CAM plasmid which degrade camphor
XYL plasmid for xylene
NAH plasmid for napthalene

Good plasmid cloning vehicles share a number of desirable features:

An ideal cloning vehicle would have the following three properties:

low molecular weight;
ability to confer readily selectable phenotypic traits on host cells;
single sites for a large number of restriction endonucleases, preferably in genes with a readily scorable phenotype.

Advantages

The advantages of a low molecular weight are several.

First, the plasmid is much easier to handle, i.e. it is more resistant to damage by shearing, and is readily isolated from host cells.
Secondly, lowmolecular-weight plasmids are usually present asmultiple copiesand this not only facilitates their isolation but leads to gene dosage effects for all cloned genes. Finally, with a low molecular weight there is less chance that the vector will have multiple substrate sites for any restriction endonuclease.

IMPORTANT VECTORS USED FOR RECOMBINANT DNA TECHNOLOGY

pBR322 vector

Pbr322 is an early example of a widely used, purpose-built cloning vector.

The best, and most widely used of these early purpose-built vectors is pBR322. Plasmid pBR322 contains the ApRandTcRgenes of RSF2124 and pSC101, respectively, combined with replication elements of pMB1, a Col E1-like plasmid.Plasmid pBR322 has been completely sequenced. The original published sequence (Sutcliffe 1979) was 4362 bp long. Position O of the sequence was arbitrarily set between the A and T residues of the EcoRI recognition sequence (GAATTC). The sequence was revised by the inclusion of an additional CG base pair at position 526, thus increasing the size of the plasmid to 4363 bp (Backman& Boyer 1983, Peden 1983). Watson (1988) later revised the size yet again, this time to 4361 bp, by eliminating base pairs at coordinates 1893 and 1915. The most useful aspect of the DNA sequence is that it totally characterizes pBR322 in terms of its restriction sites, such that the exact length of every fragment can be calculated. These fragments can serve as DNA markers for sizing any other DNA fragment in the range of several base pairs up to the entire length of the plasmid. There are over 40 enzymes with unique cleavage sites on the pBR322 genome. The target sites of 11 of these enzymes lie within the tetracycline resistant (TcR) gene, and there are sites for a further two (ClaI and HindIII) within the promoter of that gene. There are unique sites for six enzymes within the ampicillin resistant (ApR) gene. Thus, cloning in pBR322 with the aid of any one of those 19 enzymes will result in insertional inactivation of either the ApR or the TcR markers. However, cloning in the other unique sites does not permit the easy selection of recombinants, because neither of the antibiotic resistance determinants is inactivated.

Following manipulation in vitro

colicells transformed with plasmids with inserts in the TcR gene can be distinguished from those cells transformed with recircularized vector. The former are ApR and tetracycline sensitive (TcS), whereas the latter are both ApR and TcR. In practice, transformants are selected on the basis of their Ap resistance and then replica-plated onto Tc-containing media to identify those that are TcS. Cells transformed with pBR322 derivatives carrying inserts in the ApR gene can be identiﬁed more readily.Detection is based upon the ability of the β-lactamase produced by ApRcells to convert penicillin to penicilloic acid, which in turn binds iodine. Transformants are selected on rich medium containing soluble starch and Tc. When colonized plates are ﬂooded with an indicator solution of iodine and penicillin, β-lactamase-producing (ApR) colonies clear the indicator solution whereas ampicillin sensitive (ApS) colonies do not.

The PstI site in the ApR gene is particularly useful, because the 3′tetranucleotide extensions formed on digestion are ideal substrates for terminal transferase. Thus this site is excellent for cloning by the homopolymer tailing method described in the previous chapter (see p. 49). If oligo-(dG.dC) tailing is used, the PstI site is regenerated and the insert may be cut out with that enzyme.

Widely used cloning vehicle

Plasmid pBR322 has been a widely used cloning vehicle.

It has been widely used as a model system for the study of prokaryotic transcription and translation, as well as investigation of the effects of topological changes on DNA conformation.
The popularity of pBR322 is a direct result of the availability of an extensive body of information on its structure and function. This in turn is increased with each new study.

Bacteriophage lambda

The genetic organization of bacteriophage lambda favors its subjugation as a vector.

Bacteriophage λ is a genetically complex but very extensively studied virus of E. coli. The DNA of phage λ, in the form in which it is isolated from the phage particle, is a linear duplex molecule of about 48.5 kbp. The entire DNA sequence has been determined. At each end are short single-stranded 5′projections of 12 nucleotides, which are complementary in sequence and by which the DNA adopts a circular structure when it is injected into its host cell, i.e. λ DNA naturally has cohesive termini, which associate to form the cossite.Functionally related genes of phage λ are clustered together on the map, except for the two positive regulatory genes N and Q. Genes on the left of the conventional linear map.code for head and tail proteins of the phage particle. Genes of the central region are concerned with recombination (e.g. red) and the process of lysogenization, in which the circularized chromosome is inserted into its host chromosome and stably replicated along with it as a prophage. Much of this central region, including these genes, is not essential for phage growth and can be deleted or replaced without seriously impairing the infectious growth cycle. Its dispensability is crucially important, as will become apparent later, in the construction of vector derivatives of the phage. To the right of the central region are genes concerned with regulation and prophage immunity to superinfection (N, cro, cI), followed by DNA synthesis (O, P), late function regulation (Q), and host cell lysis (S, R).

Bacteriophage lambda has sophisticated control circuits

In the lytic cycle, λ transcription occurs in three temporal stages: early, middle, and late. Basically, early gene transcription establishes the lytic cycle (in competition with lysogeny), middle gene products replicate and recombine the DNA, and late gene.products package this DNA into mature phage particles. Following infection of a sensitive host, early transcription proceeds from major promoters situated immediately to the left (PL) and right (PR) of the repressor gene (cI). This transcription is subject to repression by the product of the cI gene and in a lysogen this repression is the basis of immunity to superinfecting λ. Early

in infection, transcripts from PLandPRstop at termination sites tL and tR1. The site tR2stops any transcripts that escape beyond tR1. Lambda switches from early- to middlestage transcription by anti-termination. The Ngene product, expressed from PL, directs this switch. It interacts with RNA polymerase and, antagonizing the action of host termination protein ρ, permits it to ignore the stop signals so that PL and PR transcripts extend into genes such as red, O, and Pnecessary for the middle stage. The early and middle transcripts and patterns of expression therefore overlap. The cro product, when sufﬁcient has accumulated, prevents transcription from PLand PR. The gene Qis expressed from the distal portion of the extended PR transcript and is responsible for the middle-to-late switch. This also operates by anti-termination. The Q product speciﬁcally anti-terminates the short PR transcript, extending it into the late genes, across the cohered cos region, so that many mature phage particles are ultimately produced.Both N and Qplay positive regulatory roles essential for phage growth and plaque formation; but an N−phage can produce a small plaque if the termination site tR2is removed by a small deletion termed nin (N-independent) as in λN−nin.

Two basic types of phage lambda vectors:

insertional vectors
replacement vectors

Wild-type λDNA contains several target sites for most of the commonly used restriction endonucleases and so is not itself suitable as a vector.

Insertion vectors

Derivatives of the wild-type phage have therefore been produced that either have a single target site at which foreign DNA can be inserted

Replacement vectors

They have a pair of sites deﬁning a fragment that can be removed (stuffer) and replaced by foreign DNA. Since phage λ can accommodate only about 5% more than its normal complement of DNA, vector derivatives are constructed with deletions to increase the space within the genome. The shortest λ DNA molecules that produce plaques of nearly normal size are 25% deleted. Apparently, if too much non-essential DNA is deleted from the genome, it cannot be packaged into phage particles efﬁciently. This can be turned to advantage for, if the replaceable fragment of a replacement-type vector is either removed by physical separation or effectively destroyed by treatment with a second restriction endonuclease that cuts it alone, then the deleted vector genome can give rise to plaques only if a new DNA segment is inserted into it. This amounts to positive selection for recombinant phage carrying foreign DNA. Many vector derivatives of both the insertional and replacement types were produced by several groups of researchers early in the development of recombinant DNA technology.Most of these vectors were constructed for use with EcoRI, BamHI, or HindIII, but their application could be extended to other endonucleases by the use of linker molecules.

A number of phage lambda vectors with improved properties have been described

As with plasmid vectors, improved phage-vector derivatives have been developed. There have been several aims, among which are the following.

To increase the capacity for foreign DNA fragments, preferably for fragments generated by any one of several restriction enzymes.
To devise methods for positively selecting recombinant formation. • To allow RNA probes to be conveniently prepared by transcription of the foreign DNA insert; this facilitates the screening of libraries in chromosome walking procedures. An example of a vector with this property is λZAP.
To develop vectors for the insertion of eukaryotic cDNA (p. 104) such that expression of the cDNA, in the form of a fusion polypeptide with βgalactosidase, is driven in E. coli; this form of expression vector is useful in antibody screening. An example of such a vector is λgt11.

The most recent DNA Cloning With Vector generation of λ vectors, which are based on EMBL3 and EMBL4 have a capacity for DNA of size 9–23 kb. As well as

being chi+, they have polylinkers ﬂanking the stuffer fragment to facilitate library construction. Phages with inserts can be selected on the basis of their Spi−phenotype, but there is an alternative. The vector can be digested with BamHI and EcoRI prior to ligation with foreign DNA fragments produced with BamHI. If the small BamHI–EcoRI fragments from the polylinkers are removed, the stuffer fragment will not be reincorporated.

DNA Cloning with Single-stranded DNA Vectors

M13, f1, and fd are ﬁlamentous coliphages containing a circular single-stranded DNA molecule. These coliphages have been developed as cloning vectors, for they have a number of advantages over other vectors, including the other two classes of vector for E. coli: plasmids and phage λ. However, in order to appreciate their advantages, it is essential to have a basic understanding of the biology of ﬁlamentous phages.

Filamentous Bacteriophages

Filamentous bacteriophages have a number of unique properties that make them suitable as vectors.

The phage particles have dimensions 900 nm ×9 nm and contain a single-stranded circular DNA molecule, which is 6407 (M13) or 6408 (fd) nucleotides long. The complete nucleotide sequences of fd and M13 are available and they are 97% identical. The differences consist mainly of isolated nucleotides here and there, mostly affecting the redundant bases of codons, with no blocks of sequence divergence. The ﬁlamentous phages only infect strains of enteric bacteria harboring F pili. The adsorption site appears to be the end of the F pilus, but exactly how the phage genome gets from the end of the F pilus to the inside of the cell is not known. Replication of phage DNA does not result in host-cell lysis. Rather, infected cells continue to grow and dividea slower rate than uninfected cells, and extrude virus particles. Up to 1000 phage particles may be released into the medium per cell per generation.

The single-stranded phage DNA enters the cell by a process in which decapsidation and replication are tightly coupled. The capsid proteins enter the cytoplasmic membrane as the viral DNA passes into the cell while being converted to a double-stranded replicative form (RF). The RF multiplies rapidly until about 100 RF molecules are formed inside the cell. Replication of the RF then becomes asymmetric, due to the accumulation of a viral-encoded singlestranded speciﬁc DNA-binding protein. This protein binds to the viral strand and prevents synthesis of the complementary strand. From this point on, only viral single strands are synthesized. These progeny single strands are released from the cell as ﬁlamentous particles following morphogenesis at the cell membrane. As the DNA passes through the membrane, the DNA-binding protein is stripped off and replaced with capsid protein.

Phage M13

Phage M13 has been modiﬁed to make it a better vector

Unlike λ, the ﬁlamentous coliphages do not have any non-essential genes which can be used as cloning sites. However, in M13 there is a 507 bpintergenic region, from position 5498 to 6005 of the DNA sequence, which contains the origins of DNA replication for both the viral and the complementary strands. In most of the vectors developed so far, foreign DNA has been inserted at this site, although it is possible to clone at the carboxy-terminal end of gene IV . The wild-type phages are not very promising as vectors because they contain very few unique sites within the

intergenic region: AsuI in the case of fd, and AsuI and AvaI in the case of M13. The ﬁrst example of M13 cloning made use of one of 10 BsuI sites in the genome, two of which are in the intergenic region. For cloning, M13 RF was partially digested with BsuI and linear full-length molecules isolated by agarose gel electrophoresis. These linear monomers wereblunt-end-ligated to a HindII restriction fragment comprising the E. coli lac regulatory region and the genetic information for the α-peptide of βgalactosidase. The complete ligation mixture was used to transform a strain of E. coliwith a

deletion of the β-galactosidase α-fragment and recombinant phage detected by intragenic complementation on media containing IPTG and Xgal. One of the blue plaques was selected and the virus in it designated M13 mp1. Insertion of DNA fragments into the lac region of M13 mp1 destroys its ability to form blue plaques, making detection of recombinants easy. However,

the lac region only contains unique sites for AvaII, BglI, and PvuI and three sites for PvuII, and there are no sites anywhere on the complete genome for the commonly used enzymes such as EcoRI or HindIII. To remedy this defect, Gronenborn and Messing (1978) used in vitro mutagenesis to change a single base pair,

thereby creating a unique EcoRI site within the lac fragment. This variant was designated M13 mp2. This phage derivative was further modiﬁed to generate derivatives with polylinkers upstream of the lac α-fragment.

Vectors for Cloning large Fragments of DNA:

Cosmids

Cosmids are plasmids that can be packaged into bacteriophage lambda

Particles.Concatemers of unit-length λDNA molecules can be efﬁciently packaged if the cossites, substrates for the packaging-dependent cleavage, are 37–52 kb apart (75–105% the size of λ+DNA). In fact, only a small region in the proximity of the cos site is required for recognition by the packaging system.Plasmids have been constructed which contain a fragment of λ DNA including the cos site.These plasmids have been termed cosmids and can be used as gene-cloning vectors in conjunction with the in vitro packaging system. recombinants into phage coats imposes a desirable selection upon their size. With a cosmid vector of 5 kb, we demand the insertion of 32–47 kb of foreign DNA – much more than a phage-λ vector can accommodate. Note that, after packaging in vitro, the particle is used to infect a suitable host. The recombinant cosmid DNA is injected and circularizes like phage DNA but replicates as a normal plasmid without the expression of any phage functions. Transformed cells are selected on the basis of a vector drugresistance marker.Cosmids provide an efﬁcient means of cloning large pieces of foreign DNA. Because of their capacity for large fragments of DNA, cosmids are particularly attractive vectors for constructing libraries of eukaryotic genome fragments. Partial digestion with a restriction end on nuclease provides suitably large fragments. However, there is a potential problem associated with the use of partial digests in this way. This is due to the possibility of two or more genome fragments joining together in the ligation reaction, hence creating a clone containing fragments that were not initially adjacent in the genome. Even with sized foreign DNA, it is possible for cosmid clones to be produced that contain non contiguous DNA fragments ligated to form a single insert. The problem can be solved by dephosphorylating the foreign DNA fragments so as to prevent their ligation together. This method is very sensitive to the exact ratio of target-to-vector DNAs because vector-to-vector ligation can occur. Furthermore, recombinants with a duplicated vector are unstable and break down in the host by recombination, resulting in the propagation of a non-recombinant cosmid vector.Such difﬁculties have been overcome in a cosmidcloning procedure devised by Ish-Horowicz and Burke(1981). By appropriate treatment of the cosmid vector pJB8 left-hand and right-hand vector ends are puriﬁed which are incapable of self ligation but which accept dephosphorylated foreign DNA. Thus the method eliminates the need to size the foreign DNA fragments and prevents formation of clones containing short foreign DNA or multiple vector sequences. An alternative solution to these problems has been devised by Bates and Swift (1983) who have constructed cosmid c2XB. This cosmid carries a BamHI insertion site and two cossites separated by a blunt-end restriction site . The creation of these blunt ends, which ligate only very inefﬁciently under the conditions used, effectively prevents vector self-ligation in the ligation reaction.

BACs and PACs

BACs and PACs are vectors that can carry much larger fragments of DNA than cosmids because they do not have packaging constraints. Phage P1 is a temperate bacteriophage which has been extensively used for genetic analysis of Escherichiacolibecause it can mediate generalized transduction. Sternberg and co-workers have developed a P1 vector system which has a capacity for DNA fragments as large as 100 kb .Thus the capacity is about twice that of cosmid clones but less than that of yeast artiﬁcial chromosome (YAC) clones. The P1 vector contains a packaging site (pac) which is necessary for in vitropackaging of recombinant molecules into phage particles. The vectors contain two loxP sites. These are the sites recognized by the phage recombinase, the product of the phage cre gene, and which lead to circularization of the packaged DNA after it has been injected into an E. coli host expressing the recombinase. Clones are maintained in E. colias low-copy-number plasmids by selection for

a vector kanamycin-resistance marker. A high copy number can be induced by exploitation of the P1 lytic replicon (Sternberg 1990). This P1 system has been used to construct genomic libraries of mouse, human, ﬁssion yeast, and DrosophilaDNA.

Shizuya et al. (1992) have developed a bacterial cloning system for mapping and analysis of complex genomes. This BAC system (bacterial artiﬁcial chromosome) is based on the single-copy sex factor F of E. coli. This vectorincludes the λ cos N and P1 loxP sites, two cloning sites (HindIII and BamHI), and several G+C restriction enzyme sites (e.g. SﬁI, NotI, etc.) for potential excision of the inserts. The cloning site is also ﬂanked by T7 and SP6 promoters for generating RNA probes. This BAC can betransformed into E. colivery efﬁciently, thus avoiding the packaging extracts that are required with the P1 system. BACs are capable of maintaining human and plant genomic fragments of greater than 300 kb for over 100 generations with a high degree of stabilityand have been used to construct genome libraries with an average insert size of 125 kbSubsequently, Ioannou et al. (1994) have developed a P1-derived artiﬁcial chromosome (PAC), by combining features of both the P1 and the F-factor systems. Such PAC vectors are able to handle inserts in the 100–300 kb range. The ﬁrst BAC vector, pBAC108L, lacked a selectable marker for recombinants. Thus, clones with inserts had to be identiﬁed by colony hybridization. While this once was standard practice in gene manipulation work, today it is considered to be inconvenient! Two widely used BAC vectors, pBeloBAC11 and pECBAC1, are derivatives of pBAC108L in which the original cloning site is replaced with a lacZ genecarrying a multiple cloning site (Kim et al. 1996, Frijters et al.1997). pBeloBAC11 has two EcoRI sites, one in the lacZ gene and one in the CMRgene, whereas pECBAC1 has only the EcoRI site in the lacZ gene. Further improvements to BACs have been made by replacing the lacZ gene with the sacB gene (Hamilton et al. 1996). Insertional inactivation of sacB permits growth of the host cell on sucrose-containing media, i.e. positive selection for inserts. Frengen et al.(1999) have further improved these BACs by including a site for the insertion of a transposon. This enables genomic inserts to be modiﬁed after cloning in bacteria, a procedure known as retroﬁtting. The principal uses of retroﬁtting are the simpliﬁed introduction of deletions (Chatterjee&Coren 1997) and the introduction of reporter genes for use in the original host of the genomic DNA (Wang et al. 2001). For example, Al-Hasani et al. (2003) and Magin-Lachmann et al. (2003) have used retroﬁtting to develop BACs that facilitate transfection, episomal maintenance,and functional analysis of large genomic.

Specialist-purpose vectors

M13-based vectors can be used to make single-stranded DNA suitable for sequencing.

Whenever a new gene is cloned or a novel genetic construct is made, it is usual practice to sequence all or part of the chimeric moleculethe Sanger method of sequencing requires single-stranded DNA as the starting material. Originally, single-stranded DNA was obtained by cloning the sequence of interest in an M13 vector.Today, it is more usual to clone the sequence into a pUC-based phagemid vector which contains the M13 ori region as well as the pUC (Col E1) origin of replication. Such vectors normally replicate inside the cell as double-stranded molecules. Single-stranded DNA for sequencing can be produced by infecting cultures with a helper phage such as M13K07. This helper phage has the origin of replication of P15A and a kanamycin-resistance gene inserted into the M13 oriregion and carries a mutation in the gIIgene. M13K07 can replicate on its own. However, in the presence of a phagemid bearing a wild-type origin of replication, singlestrandedphagemid is packaged preferentially and secreted into the culture medium. DNA puriﬁed from the phagemids can be used directly for sequencing.

Specialist vectors have been developed that facilitate the production of RNA probes and interfering RNA

Single-stranded DNA can be used as a sequence probe in hybridization experiments, RNA probes are preferred. The reasons for this are that the rate of hybridization and the stability are far greater for RNA–DNA hybrids compared with DNA–DNA hybrids. To make an RNA probe, the relevant gene sequence is cloned in a plasmid vector such that it is under the control of a phage promoter. After puriﬁcation, the plasmid is linearized with a suitable restriction enzyme and then incubated with the phage RNA polymerase and the four ribonucleoside triphosphates.No transcription terminator.is required because the RNA polymerase will fall off the end of the linearized plasmid. There are three reasons for using a phage promoter. First, such promoters are very strong, enabling large amounts of RNA to be made in vitro. Secondly, the phage promoter is not recognized by the E. coliRNA polymerase and so no transcription will occur inside the cell. This minimizes any selection of variant inserts. Thirdly, the RNA polymerases encoded by phages such as SP6, T7, and T3 are much simpler molecules to handle than the E. coli enzyme, since the active enzyme is a single polypeptide. If it is planned to probe RNA or single-stranded DNA sequences, then it is essential to prepare RNA probes corresponding to both strands of the insert. One way of doing this is to have two different clones corresponding to the two orientations of the insert. An alternative method is to use a cloning vector in which the insert is placed between two different, opposing phage promoters (e.g. T7/T3 or T7/SP6) that ﬂank a multiple cloning sequence (see Fig. 5.5). Since each of the two promoters is recognized by a different RNA polymerase, the direction of transcription is determined by which polymerase is used.A further improvement has been introduced byIn their LITMUS vectors, the polylinker regions are ﬂanked by two modiﬁed T7 RNA poly maser promoters. Each contains a unique restriction site (SpeI or AﬂII) that has been engineered into the T7 promoter consensus sequence such that cleavage with the corresponding endonuclease inactivates that promoter. Both promoters are active despite the presence of engineered sites. Selective unidirectional transcription is achieved by simply inactivating the other promoter by digestion with SpeI or AﬂII prior to in vitro transcription e cloned insert contain either anSpeI or an AﬂII site, the unwanted promoter can be inactivated by cutting at one of the unique sites within the poly linker. RNA interference (RNAi) is a mechanism of post-transcriptional gene silencing in which double stranded RNA corresponding to a gene of interest is introduced into an organism, thereby causingdegradation of the matching mRNA. The applications of this technique are discussed in detail on p. 318. The easiest way to make double-stranded RNA is to use vectors like the LITMUS ones just described. In this case the plasmid DNA containing the cloned target of interest is digested, in separate reactions, with SpeI and AﬂII. This will generate a template for each RNA strand. If the templates are mixed and used for in vitro transcription then double-stranded RNA will be produced.

This diagram shows the use of LITMUS VECTOR for making RNA probes as a specialist vector.

Puriﬁcation of a Cloned Gene Product can be Facilitated by use of Puriﬁcation Tags

Many cloning vectors have been engineered so that the protein being expressed will be fused to another protein, called a tag, which can be used to facilitate protein puriﬁcation. Examples of tags include glutathione-S-transferase, the MalE (maltose-binding) protein, and multiple histidine residues, which can easily be puriﬁed by afﬁnity chromatography. The tag vectors are usually constructed so that the coding sequence for an amino acid sequence cleaved by a speciﬁc protease is inserted between the coding sequence for the tag and the gene being expressed. After puriﬁcation, the tag protein can be cleaved off with the speciﬁc protease to leave a normal or nearly normal protein. It is also possible to include in the tag a protein sequence that can be assayed easily. This permits assay of the cloned gene product when its activity is not known or when the usual assay is inconvenient. To use a polyhistidine fusion for puriﬁcation, the gene of interest is ﬁrst engineered into a vector in which there is a polylinker downstream of sixhistidine residues and a proteolytic cleavage site.the cleavage site is that for enterokinase. After induction of synthesis of the fusion protein, the cells are lysed and the viscosity of the lysate is reduced by nuclease treatment. The lysate is then applied to a column containing immobilized divalent nickel, which selectively binds the polyhistidine tag. After washing away any contaminating proteins, the fusion protein is eluted from the column and treated with enterokinase to release the cloned gene product. For the cloned gene to be expressed correctly, it has to be in the correct translational reading frame. This is achieved by having three different vectors, each with a polylinker in a different reading frame.Enterokinase recognizes the sequence (Asp)4Lys and cleaves immediately after the lysine

residue. Therefore, after enterokinase cleavage, the cloned gene protein will have a few extra amino acids at the N terminus. If desired, the cleavage site and polyhistidines can be synthesized at the C terminus. If the cloned gene product itself contains an enterokinase cleavage site, then an alternative protease, such as thrombin or factor Xa, with a different cleavage site can be used. To facilitate assay of the fusion proteins, short antibody recognition sequences can be incorporated into the tag between the afﬁnity label and the protease cleavage site. Some examples of recognizable epitopes are given in Table 5.4. These antibodies can be used to detect, by western blotting, fusion proteins carrying the appropriate epitope. Note that a polyhistidine tag at the C terminus can function for both assay and puriﬁcation.Biotin is an essential cofactor for a number of carboxylases important in cell metabolism. The biotin in these enzyme complexes is covalently attached at a speciﬁc lysine residue of the biotin carboxylase carrier protein. Fusions made to a segment of the carrier protein are recognized in E. coli by biotin ligase, the product of the birA gene, and biotin is covalently attached in an ATP-dependent reaction. The expressed fusion protein can be puriﬁed using streptavidin afﬁnity chromatography . E. coli expresses a single endogenous biotinylated protein, but it does not bind to streptavidin in its native conﬁguration, making the afﬁnity puriﬁcation highly speciﬁc for the recombinant fusion protein. The presence of biotin on the fusion protein has an additional advantage: its presence can be detected with enzymes coupled to streptavidin. The afﬁnity puriﬁcation systems described above suffer from the disadvantage that a protease is required to separate the target protein from the afﬁnity tag. Also, the protease has to be separated from the protein of interest. Chong et al. (1997, 1998) have described a unique puriﬁcation system that has neither of these disadvantages. The system utilizes a protein splicing element, an intein, from the Saccharomyces cerevisiae VMA1 gene. The intein is modiﬁed such that it undergoes a self-cleavage reaction at its N terminus at low temperatures in the presence of thiols, such as cysteine, dithiothreitol, or β-mercaptoethanol. The gene encoding the target protein is inserted into a multiple cloning site (MCS) of a vector to create a fusion between the C terminus of the target gene and the N terminus of the gene encoding the intein. DNA encoding a small (5 kDa) chitin-binding domain from Bacillus circulanswas added to the C terminus of the intein for afﬁnity puriﬁcation.

The Gateway® system is a highly efﬁcient method for transferring DNA fragments to a large number of different vectors

The traditional method for moving a gene fragment from one vector to another would involve restriction enzyme digestion and would include some or all of the following steps:

Restriction endonuclease digestion of the donor plasmid;
Puriﬁcation of the gene insert;
Restriction digestion of the target vector;
Ligation of the gene insert with the digested target vector;
Transformation of E. coliand selection of the new recombinant plasmid;
Isolation of the new plasmid and conﬁrmation by endonuclease digestion and gel electrophoresis that it has the correct properties.

The Gateway system is designed to replace all these steps by using the phage λsite-speciﬁc recombinase simple two-step procedure.

To use the Gateway system, the gene of interest is cloned by conventional means in a Gateway entry vector. This vector carries two att sites that are recognized by the λsite-speciﬁc recombinase and the cloned gene should lie between them. Moving this cloned gene to another vector (destination vector) is very simple. The entry vector containing the cloned gene is mixed with the destination vector and λ recombinase in vitro and after a short incubation period the desired recombinant is selected by transformation. The beauty of the Gateway system is that after the initial entry clone is made the gene of interest can be transferred to many other vectors (Fig. 5.19) while maintaining orientation and reading frame with high efﬁciencies (>99%).

Conclusion

Many of the vectors in current use, particularly those that are commercially available, have combinations of the features.

Two examples are described here to show the connection between the different features. The ﬁrst example is the LITMUS vectors that were described earlier (p. 83) and which are used for the generation of RNA probes. They exhibit the following features:

The polylinkers are located in the lacZ′ gene and inserts in the polylinker prevent α-complementation. Thus blue/white screening can be used to distinguish clones with inserts from those containing vector only.
The LITMUS polylinkers contain a total of 32 unique restriction sites. Twenty-nine of these enzymes leave four-base overhangs and three leave blunt ends. The three blunt cutting enzymes have been placed at either end of the polylinker and in the middle of it.
The vectors carry both the pUC and the M13 ori regions. Under normal conditions the vector replicates as a double-stranded plasmid but, on infection with helper phage (M13KO7), singlestranded molecules are produced and packaged in phage protein.
The vectors are small (<3 kb) and with a pUCori have a high copy number.
Expression is under the control of either the T7 or the tacpromoter, allowing the user great ﬂexibility of control over the synthesis of the cloned gene product.
Some of them carry a DNA sequence specifying synthesis of a signal peptide. Presence of an MCS adjacent to a factor-Xa cleavage site.
Synthesis of an N-terminal biotinylated sequence to facilitate puriﬁcation.
features necessary for DNA sequencing
Three different vectors of each type permitting translation of the cloned gene insert in each of the three reading frames.
Presence of a phage SP6 promoter distal to the MCS to permit the synthesis of RNA probes complementary to the cloned gene. Note that the orientation of the cloned gene is known and so the RNA probe need only be synthesized from one strand.
What is absent from these vectors is an M13 origin of replication to facilitate synthesis of single strands for DNA sequencing.

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