Vectors for moleculars cloning
Phage M13 is widely used in nucleotide sequencing and site-directed mutagenesis
since its genome can exist either in a single-stranded form inside a phage coat
or as a doublestranded replicative form within the infected cell. During replication,
only the plus strand of the replicative form is selectively packaged by the phage
proteins [1]. The replicative form is a covalently closed circular molecule and
hence can be used as a plasmid vector and transformed into the host by the usual
transformation procedures. The vectors derived from M13, have the same polylinker
as that of pUC18 and pUC19, respectively [2]. The DNA fragments having noncomplementary
ends can be directionally cloned in this pair of vectors, and the two strands of
DNA can be sequenced independently.
4.2.Double-stranded phage vectors
Of the double-stranded phages, bacteriophage lambda-derived vectors are the
most popular tools for several reasons:
·
acceptance by the phage of large foreign
DNA fragments, thereby increasing the chances of screening a single clone carrying
a DNA sequence corresponding to a complete gene;
·
development and availability of refined
techniques aimed at minimizing the problems of background due to nonrecombinants;
·
the possibility of screening several thousand
clones at a time from a single petri plate; and, finally,
·
the ease with which the phage library can
be stored as a clear lysate at 4°C for months without significant loss in plaque-forming
activity [7].
Recently, a bacteriophage P1 cloning system has been developed which permits
cloning of DNA fragments as large as 100 kbp with an efficiency that is intermediate
between cosmids and yeast artificial chromosomes .
5.
Scope
of Present Review
The extensive knowledge of the basic biology of lambda has permitted modifications
of its genome to suit the given experimental conditions. In the present review
we describe how the utility of lambda as a cloning vector rests essentially in its
intrinsic molecular organization. The following sections give an account of various
problems encountered in constructing lambda vectors and the strategies that have
been adopted to overcome them. A few commonly used vectors are described in detail,
taking into account their special values and limitations. The different methods
for screening and storage of genomic and cDNA libraries in lambda vectors are also
discussed.
6.
Life
cycle and genetics of Lambda
An understanding of the basic biology of lambda, its mode of propagation,
and the genetic and molecular mechanisms that control its life cycle is needed
before its applications for genetic manipulations are discussed. This section deals
with the basic biology of lambda.
The lambda virus particle contains a linear DNA of 48,502 bp with a single-stranded
5' extension of 12 bases at both ends; these extensions are complementary to each
other.
These ends are called cohesive ends or cos. During infection, the right
5' extension (cosR), followed by the entire genome, enters the host cell. Both
the cos ends are ligated by E. coli DNA ligase, forming a covalently closed circular
DNA which is acted upon by the host DNA gyrase, resulting in a supercoiled structure.
6.1 Development of Lambda
Two Alternative Modes. After infecting the host, the lambda genome may start
its replication; this results in the formation of multiple copies of the genome.
The protein components necessary for the assembly of mature phage particles are
synthesized by the coordinated expression of phage genes. Phage DNA is packaged
inside a coat, and the mature phages are released into the environment after cell
lysis. This mode of propagation is called the lytic cycle.
Alternatively, the phage genome may enter a dormant stage (prophage) by integrating
itself into a bacterial genome by site-specific recombination; during this stage
it is propagated along with the host in the subsequent progeny. This stage is termed
lysogeny. Changes in environmental and physiological conditions may activate the
prophage stage and trigger lytic events.
7.
Phage
Lambda as a vector
Figure 6. Bacteriophage
The large genome size and complex genetic organization of lambda had posed
initial problems with its use as a vector. The problems, however, were surmounted
through the sustained efforts of researchers, and lambda has been developed into
an efficient vector.
The broad objectives in constructing various phage vectors are
§ the
presence of cloning sites only in the dispensable fragments,
§ the
capacity to accommodate foreign DNA fragments of various sizes,
§ the
presence of multiple cloning sites,
§ an
indication of incorporation of DNA fragments by a change in the plaque type,
§ the
ability to control transcription of a cloned fragment from promoters on the vector,
§ the
possibility of growing vectors and clones to high yield,
§ easy
and ready recovery of cloned DNA,
§ introduction
of features contributing to better biological containment.
There are several difficulties in the use of lambda as a vector.
Some of the problems and the general strategies adopted to overcome them
are discussed in this section. Manipulation of Restriction Sites The major obstacle
to the use of phage lambda as a cloning vector was essentially the presence of
multiple recognition sites for a number of restriction enzymes in its genome.
Initially, all attempts were directed toward minimizing the number of EcoRI
sites. Murray and Murray in 1974 were able to construct derivatives of lambda with
only one or two EcoRI sites. Similarly, Rambach and Toillais constructed lambda
derivatives with EcoRI sites only in the nonessential region of the genome by repeated
transfer on restrictive and nonrestrictive hosts . After several cycles of digestion,
packaging, and growth, phage derivatives with desirable restriction sites and full
retention of infectivity were obtained. All but one HindIII sites were removed
by recombination of known deletion mutants or substitutions. Recently, oligonucleotides
with specific sequences have been synthesized and introduced into the bacteriophage
lambda genome. This has provided a variety of cloning sites in the genome [5].
7.1 Size Limitation for Packaging
The second problem was the requirement of a minimum and maximum genome length
(38 and 53 kbp, respectively) for the efficient packaging and for the production
of viable phage particles. The viability of the bacteriophage decreases when its
genome length is greater than 105% or less than 78% of that of wild-type lambda.
Genetic studies of specialized transducing bacteriophages showed, however, that
the central one-third of the genome, i.e., the region between the J and Ngenes,
is not essential for lytic growth. The presence of a nonessential middle fragment
of the phage genome was also revealed during construction of viable deletion mutants.
These mutants lack most of the two central EcoRI B fragments which are not essential
for lytic growth. However, too much DNA cannot be deleted because there is a minimum
38-kbp requirement essential for efficient packaging. The de novo insertion of
DNA (even if heterogeneous) is essential for the formation of viable phages. This
constitutes a positive selection for recombinant phages carrying insertions. This
approach was successfully exploited in constructing recombinant phages carrying
E. coli and Drosophila melanogaster DNA [8].
7.2 Transfection of Recombinant Molecules
The problem of transfection of recombinant molecules constructed in vitro
was overcome by the successful in vitro assembly of viable and infectious phage
particles. Two types of in vitro packaging systems have been developed so far, i.e.,
two-strain packaging and single-strain packaging.
Two-strain packaging.
The basis of the two-strain in vitrop ackaging system is the complementation
of two amber mutations. Two lambda lysogens, each carrying a single amber mutation
in a distinctly different gene, are induced and grown separately so that they can
synthesize the necessary proteins. Neither of the lysogens alone is capable of packaging
the phage DNA. The role of various phage products in DNA packaging has been studied
in detail[3]. The E protein is the major component of the bacteriophage head, and
in its absence all the viral capsid components accumulate. The D protein is involved
in the coupled process of insertion of bacteriophage DNA into the prehead precursor
and the subsequent maturation of the head. The A protein is required for the cleavage
of the concatenated precursor DNA at the cos sites. Two phage lysogens carrying
A and E or D and E mutations in the phage genome are induced separately, and cell
extracts are prepared. Neither of the extracts can produce infectious phage particles.
However, when the extracts are mixed, mature phage particles are produced by complementation.
The major drawback of the two-strain system is the competition of native phage
DNA with recombinant molecules. In both the cell extracts, native phage DNA is also
present and can be packaged with an efficiency equal to that of the chimeric DNA.
This reduces the proportion of recombinants obtained in a library. The problem of
regeneration of endogenous phages obtained in the library was partially overcome
by the use of b2-deleted prophages, which poorly excise out of the host chromosome
or by UV irradiation of packaging extracts.
Single-strain packaging.
Rosenberg have successfully developed a single-strain packaging system by
introducing deletion in the cos region of prophage, rendering the prophage DNA unpackagable
because cos is the packaging origin. Induction of the lysogen results in the intracellular
accumulation of all protein components needed for packaging.
However, packaging of phage DNA is prevented by the lack of cos sites on
the prophage DNA. On the other hand, exogenous DNA with cos sites is packaged efficiently
to produce an infectious bacteriophage particle. The single-strain system is superior
to two-strain system in having a lower background of parental phages. In addition,
it uses E. coli C, which lacks the EcoK restriction system, as the host for the
lysogen.
7.3 Biological Containment
The biological containment of recombinant phages is an important aspect
from the point of view of ethics and eventual biohazards. It is desirable that cloning
vectors and recombinants have poor survival in the natural environment and require
special laboratory conditions for their replication and survival. According to
Blattner, the lytic phages offer a natural advantage in this respect since the phage
and the sensitive bacteria coexist only briefly. A newly inserted segment may not
be compatible with E. coli metabolism for extended periods. To make the phage vectors
more safe, three amber mutations were introduced in its genome. The new vector
Xgt WES XC is safer because an amber suppressor host strain is a very rare occurrence
in the natural environment. Many vectors carry one of the amber mutations on the
genome so that they can be propagated only on an appropriate suppressor host.
8.
Phage
vectors
Many phage vectors have been constructed in the recent past, each with its
own special features. There is no universal lambda vector which can fulfill all
the desired objectives of the cloning experiments.
The
choice of a vector depends mainly on
§ the
size of a DNA fragment to be inserted,
§ the
restriction enzymes to be used,
§ the
necessity for expression of the cloned fragment,
§ the
method of screening to be used to select the desired clones.
Bacteriophage lambda vectors can be broadly classified into two types:
1.
replacement vectors ,
2.
insertion vectors.
Figure 7. Lambda Phage genome
8.1 Replacement Vectors
Taking advantage of the maximum and minimum genome size essential for efficient
packaging and the presence of the nonessential central fragment, it is possible
to remove the stuffer fragment and replace it with a foreign DNA fragment in the
desired size range. This forms the basis of lambdaderived replacement vectors.
Cloning of a foreign DNA in these vectors involves
·
preparation of left and right arms by physical
elimination of the nonessential region,
·
ligation of the foreign DNA fragment between
the arms,
·
in vitro packaging and infection.
The replacement vectors contain a pair of restriction sites to excise the
central stuffer fragments, which can be replaced by a desired DNA sequence with
compatible ends. The presence of identical sites within the stuffer fragment but
not in the arms facilitates the separation of the arms and the stuffer on density
gradient centrifugation. In many vectors, sets of such sites are provided on attached
polylinkers so that an insert can be easily excised. Two purified arms cannot be
packaged despite their being ligated to each other, because they fall short of
the minimum length required for packaging. This provides positive selection of
recombinants. The replacement vectors are convenient for cloning of large (in some
cases up to 24 kbp) DNA fragments and are useful in the construction of genomic
libraries of higher eukaryotes. Charon and EMBL are among the popular replacement
vectors.
8.2 Insertion Vectors
Because the maximum packagable size of lambda genome is 53 kb, small DNA
fragments can be introduced without removal of the nonessential (stuffer) fragment.
These vectors are therefore called insertion vectors. Cloning of foreign DNA in
these vectors exploits the insertional inactivation of the biological function,
which differentiates recombinants from nonrecombinants. Insertion vectors are particularly
useful in cloning of small DNA fragments such as cDNA.
AgtlO and Agtll are examples of this type of vector. In recent years a multitude
of lambda vectors have been constructed. Many innovative approaches have been used
to introduce desired properties into the vectors. The following section deals with
the strategies adopted for the construction of some of the commonly used vectors
and their salient features, utilities, and limitations.
8.3 Storage of Lambda Stocks
Most of the lambda strains are stable for several years when stored at 4°C
in SM buffer containing 0.3% freshly distilled chloroform (94). The master stocks
of bacteriophage lambda are kept in 0.7% (vol/vol) dimethyl sulfoxide at -70°C
for long-term storage. Klinman and Cohen have developed a method for storage of
a phage library at -70°C by using top agar containing 30% glycerol.
Conclusion
In my work I determined
investigations in Molecular cloning, familiarized with vectors for molecular cloning,
summarized the received information and made consequences of scientists researches,
defined the main tasks of molecular cloning, and made such conclusions:
1.
sequences
that permit the propagation of itself in bacteria (or in yeast for YACs) .
2.
a cloning
site to insert foreign DNA; the most versatile vectors contain a site that can
be cut by many restriction enzymes .
3.
a method
of selecting for bacteria (or yeast for YACs) containing a vector with foreign
DNA; uually accomplished by selectable markers for drug resistance .
Cloning vector - a DNA molecule that carries
foreign DNA into a host cell, replicates inside a bacterial (or yeast) cell and
produces many copies of itself and the foreign DNA .
General Steps of
Cloning with Any Vector :
1.
prepare
the vector and DNA to be cloned by digestion with restriction enzymes to generate
complementary ends ;
2.
ligate
the foreign DNA into the vector with the enzyme DNA ligase;
3.
introduce
the DNA into bacterial cells (or yeast cells for YACs) by transformation ;
4.
select
cells containing foreign DNA by screening for selectable markers (usually drug
resistance);
Literature
1.
Finbar Hayes The Function and Organization
of Plasmids// E. coli Plasmid Vectors Methods and Applications.- 2007.- vol.235
– pp. 1-18.
2.
Mallory J. A. White and Wade A. Nichols Cosmid
Packaging and Infection of E. coli// E. coli Plasmid Vectors Methods and Applications.-
2007.- vol.235 – pp. 67-70
3.
Tim S. Poulsen and Hans E. Johnsen BAC
End Sequencing // Bacterial Artificial Chromosomes Volume 1: Library Construction,
Physical Mapping, and Sequencing.- 2007. – vol.255 - pp.157-162.
4.
Andrew Preston Choosing a Cloning Vector//
E. coli Plasmid Vectors Methods and Applications.- 2007.- vol.235 – pp.
19-22.
5.
Sambrook, J., Fritsch, E. F., and Maniatis,
T. (eds.) (1989) Bacteriophage λvectors, in Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp.
2.3–2.125.
6.
Srividya Swaminathan and Shyam K. Sharan
Bacterial Artificial Chromosome Engineering// Bacterial Artificial Chromosomes
Volume 2 :Functional Studies.- 2007. – vol.256 – pp. 089-106
7.
www.Microbiologybytes.com
8.
www.wikigenes.org
9.
http:// plasmid.hms.harvard.edu
10.
www. Bookrags.com/YAC
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