Plasmids are commonly used as cloning vectors. They are circular DNA molecules that can replicate independently of the bacterial chromosome. Many plasmid cloning vectors were constructed from bacterial plasmids and contain an origin of replication, selectable marker like antibiotic resistance, and a multiple cloning site to insert DNA fragments. Successful clones can be identified by techniques like blue-white screening where expression of a gene like lacZ allows colonies to be screened blue or white.

1. Plasmid as cloning vector
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1. INTRODUCTION
Many important cloning vectors are derived from naturally occurring plasmid.Plasmids are
circular DNA molecules that are maintained as an episome,or extrachromosomal DNA
molecules,inside a cell.The plasmid must contain a DNA sequence that serves as an origin of
replication (ori) so that the plasmid DNA is propagated as the cell undergoes the cell division
cycle.Some plasmids contain genes that encode proteins that involved in plasmid DNA
replication.plasmid partitioning to daughter cells during cell division and self-transmissibility
from one cell to another(conjugation).Plasmid may also encode proteins that confer functions
beneficial to the host cell,such as resistance to antibiotics or to heavy metals.cloning vectors used
in bacteria typically have been constructed using DNA from several different source to provide
the most convenience to the experimenter.Cloning vectors used in yeast cells are either derived
from natural plasmids or constructed from DNA elements taken from the yeast
chromosomes,while many plasmids used in mammalian cells are derived from viruses. Many
naturally occurring plasmids contain genes that provide some benefit to the host cell, fulfilling
the plasmid’s portion of the symbiotic relationship. For example, some bacterial plasmids encode
enzymes that inactivate antibiotics. Such drug-resistance plasmids have become a major problem
in the treatment of a number of common bacterial pathogens. As antibiotic use became
widespread, plasmids containing several drug-resistance genes evolved, making their host cells
resistant to a variety of different antibiotics simultaneously. Many of these plasmids also contain
“transfer genes” encoding proteins that can form a macromolecular tube, or pilus, through which
a copy of the plasmid can be transferred to other host cells of the same or related bacterial
species. Such transfer can result in the rapid spread of drug-resistance plasmids, expanding the
number of antibiotic-resistant bacteria in an environment such as a hospital. Coping with the
spread of drug-resistance plasmids is an important challenge for modern medicine. The plasmids
most commonly used in recombinant DNA technology replicate in E. coli.Generally, these
plasmids have been engineered to optimize their use as vectors in DNA cloning. For instance, to
simplify working with plasmids, their length is reduced; many plasmid vectors are only ≈3kb in
length, which is much shorter than in naturally occurring E. coli plasmids. (The circumference of
plasmids usually is referred to as their “length,” even though plasmids are almost always circular
DNA molecules.) Most plasmid vectors contain little more than the
essential nucleotide sequences required for their use in DNA cloning: a replication origin, a
drug-resistance gene, and a region in which exogenous DNA fragments can be inserted.The
replication origin and associated control elements in a plasmid are referred to as a replicon.Many
different vectors may carry the same replicon and thus have the same or similar dna replication
mechanism.
Cloning 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 (e.g.- plasmid,
cosmid, Lambda phages).
vector containing foreign DNA is termed recombinant DNA. The four major types of vectors
are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly
used vectors are plasmids. Common to all engineered vectors are an origin of replication,
a multicloning site, and a selectable marker.

2. Plasmid as cloning vector
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The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger
sequence that serves as the "backbone" of the vector. The purpose of a vector which transfers
genetic information to another cell is typically to isolate, multiply, or express the insert in the
target cell. All vectors may be used for cloning and are therefore cloning vectors, but there are
also vectors designed specially for cloning, while others may be designed specifically for other
purposes, such as transcription and protein expression. Vectors designed specifically for the
expression of the transgene in the target cell are called expression vectors, and generally have
a promoter sequence that drives expression of the transgene. Simpler vectors called transcription
vectors are only capable of being transcribed but not translated: they can be replicated in a target
cell but not expressed, unlike expression vectors. Transcription vectors are used to amplify their
insert.
The manipulation of DNA is normally conducted on E. coli vectors, which contain elements
necessary for their maintenance in E. coli. However, vectors may also have elements that allow
them to be maintained in another organism such as yeast, plant or mammalian cells, and these
vectors are called shuttle vectors. Such vectors have bacterial or viral elements which may be
transferred to the non-bacterial host organism, however other vectors termed intragenic vectors
have also been developed to avoid the transfer of any genetic material from an alien species.[1]
Insertion of a vector into the target cell is usually called transformation for bacterial
cells, transfection for eukaryotic cells, although insertion of a viral vector is often
called transduction.
2.FEATURES OF A CLONING VECTOR
All commonly used cloning vectors in molecular biology have key features necessary for their
function, such as a suitable cloning site and selectable marker. Others may have additional
features specific to their use. For reason of ease and convenience, cloning is often performed
using E. coli. Thus, the cloning vectors used often have elements necessary for their propagation
and maintenance in E. coli, such as a functional origin of replication (ori). The ColE1 origin of
replication is found in many plasmids. Some vectors also include elements that allow them to be
maintained in another organism in addition to E. coli, and these vectors are called shuttle vector.
2.1 Cloning site
All cloning vectors have features that allow a gene to be conveniently inserted into the vector or
removed from it. This may be a multiple cloning site (MCS) or polylinker, which contains many
unique restriction sites. The restriction sites in the MCS are first cleaved by restriction enzymes,
then a PCR-amplified target gene also digested with the same enzymes is ligated into the vectors
using DNA ligase. The target DNA sequence can be inserted into the vector in a specific
direction if so desired. The restriction sites may be further used for sub-cloning into another
vector if necessary.
Other cloning vectors may use topoisomerase instead of ligase and cloning may be done more
rapidly without the need for restriction digest of the vector or insert. In this TOPO
cloning method a linearized vector is activated by attaching topoisomerase I to its ends, and this
"TOPO-activated" vector may then accept a PCR product by ligating both the 5' ends of the PCR

3. Plasmid as cloning vector
3
product, releasing the topoisomerase and forming a circular vector in the process.Another
method of cloning without the use of DNA digest and ligase is by DNA recombination, for
example as used in the Gateway cloning system.The gene, once cloned into the cloning vector
(called entry clone in this method), may be conveniently introduced into a variety of expression
vectors by recombination.
2.2 Selectable marker
A selectable marker is carried by the vector to allow the selection of
positively transformed cells. Antibiotic resistance is often used as marker, an example being
the beta-lactamase gene, which confers resistance to the penicillin group of beta-lactam
antibiotics like ampicillin. Some vectors contain two selectable markers, for example the plasmid
pACYC177 has both ampicillin and kanamycin resistance gene.[6] Shuttle vector which is
designed to be maintained in two different organisms may also require two selectable markers,
although some selectable markers such as resistance to zeocin and hygromycin B are effective in
different cell types. Auxotrophic selection markers that allow an auxotrophic organism to grow
in minimal growth medium may also be used; examples of these are LEU2 and URA3 which are
used with their corresponding auxotrophic strains of yeast.[7]
Another kind of selectable marker allows for the positive selection of plasmid with cloned gene.
This may involve the use of a gene lethal to the host cells, such as barnase,[8] Ccda,[9] and
the parD/parE toxins.[10][11] This typically works by disrupting or removing the lethal gene during
the cloning process, and unsuccessful clones where the lethal gene still remains intact would kill
the host cells, therefore only successful clones are selected.
2.3 Reporter gene
Reporter genes are used in some cloning vectors to facilitate the screening of successful clones
by using features of these genes that allow successful clone to be easily identified. Such features
present in cloning vectors may be the lacZα fragment for α complementation in blue-white
selection, and/or marker gene or reporter genes in frame with and flanking the MCS to facilitate
the production of fusion proteins. Examples of fusion partners that may be used for screening are
the green fluorescent protein (GFP) and luciferase.
2.4 Elements for expression
A cloning vector need not contain suitable elements for the expression of a cloned target gene,
such as a promoter and ribosomal binding site (RBS), many however do, and may then work as
an expression vector. The target DNA may be inserted into a site that is under the control of a
particular promoter necessary for the expression of the target gene in the chosen host. Where the
promoter is present, the expression of the gene is preferably tightly controlled and inducible so
that proteins are only produced when required. Some commonly used promoters are
the T7 and lac promoters. The presence of a promoter is necessary when screening techniques
such as blue-white selection are used.

4. Plasmid as cloning vector
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3.TYPES OF CLONING VECTORS
A large number of cloning vectors are available, and choosing the vector may depend a number
of factors, such as the size of the insert, copy number and cloning method. Large insert may not
be stably maintained in a general cloning vector, especially for those with a high copy number,
therefore cloning large fragments may require more specialized cloning vector.
 Plasmid
 Bacteriophage
 Cosmid
 Bacterial Artificial Chromosome
 Yeast Artificial Chromosome
 Human Artificial Chromosome
Figure: Plasmid PBR322
 It is isolated from E.coli
 Size: 4361 bp
 Cloning limit: 0.1-10 kb
 Marker gene: Ampicillin and Tetracycline resistant gene
 Restriction site for various restriction endonucleases.

5. Plasmid as cloning vector
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5. General Types of Plasmids
5.1 Conjugative and Non-Conjugative
There are many ways to classify plasmids from general to specific. One way is by grouping them
as either conjugative or non-conjugative. Bacteria reproduce by sexual conjugation, which is the
transfer of genetic material from one bacterial cell to another, either through direct contact or a
bridge between the two cells. Some plasmids contain genes called transfer genes that facilitate
the beginning of conjugation. Non-conjugative plasmids cannot start the conjugation process,
and they can only be transferred through sexual conjugation with the help of conjugative
plasmids.
5.2 Incompatibility
Another plasmid classification is by incompatibility group. In a bacterium, different plasmids can
only co-occur if they are compatible with each other. An incompatible plasmid will be expelled
from the bacterial cell. Plasmids are incompatible if they have the same reproduction strategy in
the cell; this allows the plasmids to inhabit a certain territory within it without other plasmids
interfering.
6.Specific Types of Plasmids
There are five main types of plasmids: fertility F-plasmids, resistance plasmids, virulence
plasmids, degradative plasmids, and Col plasmids.
6.1 Fertility F-plasmids
Fertility plasmids, also known as F-plasmids, contain transfer genes that allow genes to be
transferred from one bacteria to another through conjugation. These make up the broad category
of conjugative plasmids. F-plasmids are episomes, which are plasmids that can be inserted into
chromosomal DNA. Bacteria that have the F-plasmid are known as F positive (F+), and bacteria
without it are F negative (F–). When an F+ bacterium conjugates with an F– bacterium, two
F+ bacterium result. There can only be one F-plasmid in each bacterium.
6.2 Resistance Plasmids
Resistance or R plasmids contain genes that help a bacterial cell defend against environmental
factors such as poisons or antibiotics. Some resistance plasmids can transfer themselves through
conjugation. When this happens, a strain of bacteria can become resistant to antibiotics.
Recently, the type bacterium that causes the sexually transmitted infection gonorrhea has become
so resistant to a class of antibiotics called quinolones that a new class of antibiotics, called
cephalosporins, has started to be recommended by the World Health Organization instead. The

6. Plasmid as cloning vector
6
bacteria may even become resistant to these antibiotics within five years. According to NPR,
overuse of antibiotics to treat other infections, like urinary tract infections, may lead to the
proliferation of drug-resistant strains.
6.3 Virulence Plasmids
When a virulence plasmid is inside a bacterium, it turns that bacterium into a pathogen, which is
an agent of disease. Bacteria that cause disease can be easily spread and replicated among
affected individuals. The bacterium Escherichia coli (E. coli) has several virulence plasmids. E.
coli is found naturally in the human gut and in other animals, but certain strains of E. coli can
cause severe diarrhea and vomiting. Salmonella enterica is another bacterium that contains
virulence plasmids.
6.4 Degradative Plasmids
Degradative plasmids help the host bacterium to digest compounds that are not commonly found
in nature, such as camphor, xylene, toluene, and salicylic acid. These plasmids contain genes for
special enzymes that break down specific compounds. Degradative plasmids are conjugative.
6.5 Col Plasmids
Col plasmids contain genes that make bacteriocins (also known as colicins), which are proteins
that kill other bacteria and thus defend the host bacterium. Bacteriocins are found in many types
of bacteria including E. coli, which gets them from the plasmid ColE1.
RECOMBINANT DNA TECHNOLOGY
Recombinant DNA technology, joining together of DNA molecules from two different species
that are inserted into a host organism to produce new genetic combinations that are of value to
science, medicine, agriculture, and industry. Since the focus of all genetics is the gene, the
fundamental goal of laboratory geneticists is to isolate, characterize, and manipulate genes.
Although it is relatively easy to isolate a sample of DNA from a collection of cells, finding a
specific gene within this DNA sample can be compared to finding a needle in a haystack.
Consider the fact that each human cell contains approximately 2 metres (6 feet) of DNA.
Therefore, a small tissue sample will contain many kilometres of DNA. However, recombinant
DNA technology has made it possible to isolate one gene or any other segment of DNA,
enabling researchers to determine its nucleotidesequence, study its transcripts, mutate it in highly
specific ways, and reinsert the modified sequence into a living organism.
DNA Cloning
In biology a clone is a group of individual cells or organisms descended from one progenitor.
This means that the members of a clone are genetically identical, because cell replication

7. Plasmid as cloning vector
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produces identical daughter cells each time. The use of the word clone has been extended to
recombinant DNA technology, which has provided scientists with the ability to produce many
copies of a single fragment of DNA, such as a gene, creating identical copies that constitute a
DNA clone. In practice the procedure is carried out by inserting a DNA fragment into a small
DNA molecule and then allowing this molecule to replicate inside a simple living cell such as a
bacterium. The small replicating molecule is called a DNA vector (carrier). The most commonly
used vectors are plasmids (circular DNA molecules that originated from bacteria), viruses,
and yeast cells. Plasmids are not a part of the main cellular genome, but they can carry genes that
provide the host cell with useful properties, such as drug resistance, mating ability, and toxin
production. They are small enough to be conveniently manipulated experimentally, and,
furthermore, they will carry extra DNA that is spliced into them
Steps involved in the engineering of a recombinant DNA molecule Encyclopædia Britannic

8. Plasmid as cloning vector
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SELECTION OF TRANSFORMATION (BLUE WHITE SELECTION)
The process of colony selection can be simplified by choosing a vector and E. coli strain that are
compatible with blue/white colony screening. E. coli strains are described as having a lacZΔ
when they carry a mutation that deletes part of the β-galactosidase (lacZ) gene. The remaining
portion of the gene is called the ω-fragment. By using a plasmid that contains the deleted portion,
or α-fragment, the function of the β-galactosidase gene can be restored once the plasmid has
been incorporated into the bacterium. For blue/white colony screening, the plasmids have a
multiple cloning region within the coding sequence of the α-fragment. When a sequence is
inserted into this cloning region, the reading frame is disrupted, and a non-functional α-fragment
is produced. This fragment is incapable of α-complementation. Growing the transformed bacteria
on a plate containing 5-bromo-4-chloro-3-indoyl-β -D-galactopyranosidase (X-gal) will allow
you to distinguish between bacterial colonies formed from cells that contain plasmid with insert
from those containing plasmid without insert. Any colony containing the plasmid (and therefore
the functioning β-galactosidase gene) will turn blue, a result of the β-galactosidase activity. This
is called α-complementation. Those colonies containing plasmids with an insert can be
differentiated from those without an insert by the color of the colony (white versus blue). The
insert disrupted the β-galactosidase gene, and therefore these colonies remain white. Colonies
that did not pick up any plasmid at all will also appear as white colonies; however, most
plasmids contain an antibiotic resistance gene that can be used for selection (see below).
There are a number of strains including JM109, DH5α and XL-1 Blue that have the necessary
deletions and can be used for blue/white colony screening. However, the mechanism for
blue/white screening is slightly different for JM109 and XL-Blue. Both of these strains also have
a second mutation, laclq, which increases production of the lacl repressor that stops transcription
from the lac operon , and thus production of the α-fragment, until a substrate is present. The
substrate, the non-cleavable lactose analog, isopropyl-β-D-thiogalactopyranoside (IPTG),
relieves the repression of the lac operon and allows transcription to occur. These strains will need
to be grown on media containing IPTG as well as X-gal.
Figure 1: A schematic representation of a typical plasmid vector that can be used for blue-white screening.

9. Plasmid as cloning vector
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SCHEMATIC REPRESENTATION:
Figure 2: A schematic representation of a typical blue-white screening procedure.
Occasionally, colonies will appear pale blue, not white. As long as you see colonies on your
plate that are darker blue, try picking some of the pale blue colonies, chances are good that they
have the constructed plasmid that contains your DNA fragment.

10. Plasmid as cloning vector
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In addition to the β-galactosidase marker, most cloning plasmids will also contain a gene that
confers resistance to an antibiotic such as ampicillin. Using Ampicillin (or other appropriate
antibiotic) in your growth medium should prevent bacteria that did not take up the plasmid
during the transformation from growing. This way you can be fairly confident that the white
colonies you see on your screening plate contain plasmid with insert.
And of course it is always a good idea to run controls with your cloning experiment. A plasmid-
only control should give you a plate of blue colonies, and this will let you know that your
transformation worked. To make sure that the antibiotic in your selective medium is effective,
plate some untransformed cells. Few, if any, colonies should be observed on this plate.
LIMITATIONS OF BLUE-WHITE SCREENING
 The blue-white technique is only a screening procedure; it is not a selection technique.
 The lacZ gene in the vector may sometimes be non-functional and may not produce β-
galactosidase. The resulting colony will not be recombinant but will appear white.
 Even if a small sequence of foreign DNA may be inserted into MCS and change the
reading frame of lacZ gene. This results in false positive white colonies.
 Small inserts within the reading frame of lacZ may produce ambiguous light blue
colonies as β-galactosidase is only partially inactivated.
8.Plasmid preparation
A plasmid preparation is a method of DNA extraction and purification for plasmid DNA.
Many methods have been developed to purify plasmid DNA from bacteria. These methods
invariably involve three steps:
 Growth of the bacterial culture
 Harvesting and lysis of the bacteria
 Purification of plasmid DNA
8.1 Growth of the bacterial culture
Plasmids are almost always purified from liquid bacteria cultures, usually E. coli, which have
been transformed and isolated. Virtually all plasmid vectors in common use encode one or
more antibiotic resistance genes as a selectable marker, for example a gene encoding ampicillin
or kanamycin resistance, which allows bacteria that have been successfully transformed to
multiply uninhibited. Bacteria that have not taken up the plasmid vector are assumed to lack the
resistance gene, and thus only colonies representing successful transformations are expected to
grow. Bacteria are grown under favourable conditions.

11. Plasmid as cloning vector
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8.2 Harvesting and lysis of the bacteria
When bacteria are lysed under alkaline conditions (pH 12.0–12.5) both chromosomal DNA
and protein are denatured; the plasmid DNA however, remains stable. Some scientists reduce the
concentration of NaOH used to 0.1M in order to reduce the occurrence of ssDNA. After the
addition of acetate-containing neutralization buffer the large and less supercoiled chromosomal
DNA and proteins precipitate, but the small bacterial DNA plasmids stay in solution.
8.3 Purification of plasmid DNA
Kits are available from varying manufacturers to purify plasmid DNA, which are named by size
of bacterial culture and corresponding plasmid yield. In increasing order, these are the miniprep,
midiprep, maxiprep, megaprep, and gigaprep. The plasmid DNA yield will vary depending on
the plasmid copy number, type and size, the bacterial strain, the growth conditions, and the kit.
8.4 Minipreparation
Minipreparation of plasmid DNA is a rapid, small-scale isolation of plasmid DNA from bacteria.
It is based on the alkaline lysis method. The extracted plasmid DNA resulting from performing a
miniprep is itself often called a "miniprep". Minipreps are used in the process of molecular
cloning to analyze bacterial clones. A typical plasmid DNA yield of a miniprep is 50 to 100 µg
depending on the cell strain. Miniprep of large number of plasmids can also be done
conveniently on filter paper by lysing the cell and eluting the plasmid on to filter paper.

12. Plasmid as cloning vector
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9. SEVERAL PLASMID WITH THEIR RESTRICTION SITE:
Figure : pSS2 plasmid Figure :pCML 15 plasmid

13. Plasmid as cloning vector
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Figure :pUC19 plasmid Figure :pUC19 plasmid
10. General procedure for cloning a DNA fragment in a plasmid vector:

14. Plasmid as cloning vector
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Figure : General procedure for cloning a DNA fragment in a plasmid vector
Functions of Plasmids
The main functions of plasmids include:
 They help in providing resistance against antibiotics.
 Plasmids also help in process of fertility by helping bacteria in conjugation and other
processes.
 Again they do help in resistance but through another way, through the synthesis of toxic
substances which can kill harmful bacteria.
 Degradation is also done by plasmids, which can help in metabolic process of not suitable
molecules.
 Main function of plasmid is Virulence factor.
 In genetic engineering, vectors are nothing else than plasmids.
 Protein is also produced from plasmids through various methods.
 There are diseases which are only treated through gene therapy; in such conditions
plasmids are also required.
 In past history, plasmids can help in treating disease by helping in making of models of
disease.
 They are also used as episomes.
CONSTRUCTION OF DISARMED SHUTTLE TiPLASMIDS:
We designed a simple engineering scheme that can make pathogenic Ti plasmids disarmed,
stably maintainable in E. coli, and mobilizable between E. coli and Agrobacterium species. As an
example, we used the scheme with nopaline-type plasmids. We first constructed pLRS-GmsacB
and pLRS-Gms2 as tool plasmids to modify nopaline-type Ti plasmids. These tool plasmids are
pK18mobsacB containing two fragments, LL and RR, which neighbor to the left of LB and to
the right of RB of T-DNA in pTi-SAKURA, respectively, and a cassette containing a gentamicin
resistance gene, the low-copy-number type replication origin (oriV) derived from pSC101, and
the IncP-type transfer origin (oriT) sandwiched between LL and RR. The pSC101
replication ori should allow the chimeric plasmids to replicate at a very low copy number in E.
coli.Two nopaline-type Ti plasmids, pTiC58 and pTi-SAKURA, were modified using pLRS-
GmsacB.First, the pLRS-GmsacB plasmid in E. coli was introduced by conjugation into two
pathogenic nopaline-type strains belonging to A. tumefaciens (biovar 1). C58rif is a pathogenic
strain harboring pTiC58. C58C1 is a Ti-less strain. C58C1 harboring pTi-SAKURA is another
pathogenic strain. Because pLRS-GmsacB cannot replicate in Agrobacterium cells, the tool
plasmid should integrate into the Ti plasmids by homologous recombination at either LL or RR
in the transformants.The Agrobacterium transconjugants were resistant to gentamicin and
kanamycin and sensitive to sucrose due to the Gmr, Kmr, and sacB genes on the fusion plasmids.

15. Plasmid as cloning vector
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Figure : Conversion of pathogenic Ti plasmids so that they are disarmed and transferable
between E. coli and Agrobacterium. The modification of pTiC58 and pTi-SAKURA consists of
two steps. (A) pLRS-GmsacB was inserted in vivo into pTiC58 and pTi-SAKURA by
homologous recombination at either RR or LL.(B) Cells harboring the fused plasmid DNA were
cultivated on LB agar containing sucrose and gentamicin in order to select for the subsequent
crossover products. Only the recombinant that did not include the T-DNA portion was selected
by cultivation on the medium.
Next, the transconjugants harboring the resulting fusion plasmid were cultured on LB agar
supplemented with gentamicin and sucrose. Cultivation in a sucrose-containing medium selects
for cells that do not have the sacB gene. Loss of the fusion plasmid can occur at a high
frequency. Loss of this plasmid converts cells to Gms, Kms, sucrose-resistant cells. Deletion of
the sacB gene from the plasmid can take place at a high frequency through homologous
recombination in two ways: recombination between two RR segments, resulting in removal of
the pLRS-GmsacB portion, or, alternatively, recombination between two LL segments, resulting
in loss of the T-DNA region.The former recombination converts cells to Gms, whereas the latter
maintains Gmr genes. Thus, colonies on the selective agar plate were expected to have a
disarmed type of pTi. To confirm the lack of T-DNA in the derivatives of pTiC58 and pTi-
SAKURA, for each Ti plasmid four colonies were randomly chosen from the selective agar
culture and analyzed by PCR. T-DNA products were not detected in any of the colonies
examined, whereas the virB gene was detected in every colony examined in another PCR
experiment (data not shown). These results suggest that there was accurate and frequent removal
of the long T-DNA region by replacement using pLRS-GmsacB and the simple selection media.
The resultant Ti plasmids were designated pTiC58-S and pTi-SAKURA-S.

16. Plasmid as cloning vector
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11. Agrobacterium mediated gene transfer
Figure : Agrobacterium mediated gene transferusing Ti plasmid
11. Why Plasmids are Good Cloning Vectors:
• Small size (easy to manipulate and isolate).
• Circular (more stable).
• Replication independent of host cell.
• Several copies may be present (facilitates replication).
• Frequently have antibiotic resistance (detection easy).
12. Disadvantages of Using Plasmids:
• Cannot accept large fragments.
• Sizes range from 0 – 10kb.
• Standard methods of transformation are inefficient.
13. Conclusion:
There are different types of cloning vectors used in Genetic Engineering. The best vector is
chosen for use according to the purpose of use and according to how large/short the DNA
fragment to be carried. Despite of some limitations plasmid become most popular cloning vector.

17. Plasmid as cloning vector
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14. References:
1. Watson, N. (1988). "A new revision of the sequence of plasmid pBR322". Gene. 70(2):
399–403. doi:10.1016/0378-1119(88)90212-0. PMID 3063608.
2. Balbás P, Soberón X, Merino E, Zurita M, Lomeli H, Valle F, Flores N, Bolivar F (1986).
"Plasmid vector pBR322 and its special-purpose derivatives--a review". Gene. 50 (1-3):
3–40. doi:10.1016/0378-1119(86)90307-0. PMID 3034735.
3. "pBR322 Nucleotide Sequences, NCBI Sequence Viewer v2.0".
4. R.W. Old & S.B. Primrose. Principles of Gene Manipulation (5th ed.). pp. 53–61.
5. Manen D, Caro L (February 1991). "The replication of plasmid pSC101". Mol.
Microbiol. 5 (2): 233–7. doi:10.1111/j.1365-2958.1991.tb02103.x. PMID 2041467.
6. Bolivar F, Rodriguez RL, Betlach MC, Boyer HW (1977). "Construction and
characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the
plasmid pMB9". Gene. 2 (2): 75–93. doi:10.1016/0378-1119(77)90074-9. PMID 344136.
7. Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH,
Falkow S (1977). "Construction and characterization of new cloning vehicles. II. A
multipurpose cloning system". Gene. 2 (2): 95–113. doi:10.1016/0378-1119(77)90000-
2. PMID 344137.
8. S.B. Primrose & R.M Twyman (17 January 2006). Principles of Gene Manipulation and
Genomics (PDF) (7th ed.). Wiley-Blackwell. pp. 64–65. ISBN 978-1405135443.
9. Balbás P, Soberón X, Merino E, Zurita M, Lomeli H, Valle F, Flores N, Bolivar F (1986).
"Plasmid vector pBR322 and its special-purpose derivatives--a review". Gene. 50 (1-3):
3–40. doi:10.1016/0378-1119(86)90307-0. PMID 3034735.
10. Yanisch-Perron C, Vieira J, Messing J (1985). "Improved M13 phage cloning vectors and
host strains: nucleotide sequences of the M13mp18 and pUC19 vectors". Gene. 33 (1):
103–19. doi:10.1016/0378-1119(85)90120-9. PMID 2985470.
11. Paulina Balbás; Argelia Lorence, eds. (April 2004). Recombinant Gene Expression:
Reviews and Protocols (2nd ed.). Humana Press Inc. pp. 77–85. ISBN 978-1592597741.
12. www.chemistrylearning.com/cloning-vector/
13. www.thebalance.com/gene-cloning-and-vectors-definition-and-major-types-375681
14. www.ndsu.edu/pubweb/~mcclean/plsc431/cloning/clone3.htm
15. en.wikipedia.org/wiki/Cloning_vector