INTRODUCTION OF PLASMIDS, THEIR

IMPORTANCE AND TYPES

Plasmids are transferable extrachromosomal DNA

molecules capable of autonomous replication. They are

double stranded and mostly circular. It usually occurs

naturally in bacteria and is some times found in

eukaryotic organisms e.g. Saccharomyces cerevisiae. The

size of plasmids varies from 1 to over 400 kilo base pairs

(kbp). There may be one copy, for large plasmids, to

hundreds of copies of the same plasmid in a single cell,

or even thousands of copies, for certain artificial plasmids

selected for high copy number such as the pUC series of

plasmids. Every plasmid contains at least one DNA

sequence that serves as an origin of replication, or ori (a

starting point for DNA replication), which enables the

plasmid DNA to be duplicated independently from the

chromosomal DNA. The plasmids of most bacteria are

circular, but linear plasmids are also known, which

superficially resemble the chromosomes of most

eukaryotes.

Frequently plasmids contain some genes advantageous

to the bacterial host for example, resistance to

antibiotics, production of antibiotics and degradation of

complex organic compounds. Antibiotics are antimicrobial

agents that can be classified based on their mode of

action. Bacterial antibiotic-resistance genes encode

proteins that inactivate these agents either by preventing

their accumulation in the cell or by inactivating them

once they have been imported. By utilizing these

antibiotic-resistance genes as dominant genetic markers

in plasmid cloning vectors, it is possible to select for E.

coli cells that have maintained high copy replication of

plasmid DNA molecules.

Plasmids used in genetic engineering are called vectors.

They are used to transfer genes from one organism to

another and typically contain a genetic marker conferring

a phenotype that can be selected for or against. Plasmids

serve as important tools in genetics and biochemistry

labs, where they are commonly used to multiply (make

many copies of) or express particular genes. Many

plasmids are commercially available for such uses.

Another major use of plasmids is to make large amounts

of proteins. In this case, researchers grow bacteria

containing a plasmid harboring the gene of interest. Just

as the bacteria produce proteins to confer its antibiotic

resistance; it can also be induced to produce large

amounts of proteins from the inserted gene. This is a

cheap and easy way of mass-producing a gene or the

protein it then codes for, for example, insulin or even

antibiotics.

Some important plasmid vectors

pBR322

This was one of the first artificial cloning vectors to be

constructed, and is undoubtedly the most widely used

cloning vector till now (Bolivar and Rodriguez, 1977a;

1977b). It is a 4.36-kb double stranded cloning vector.

This plasmid vector has been put together from

fragments originating from three different naturally

occurring plasmids. Figure 1a indicates all the salient

features of this vector. It contains CoIE1 ori of

replication with relaxed replication control. Generally

there are 15-20 molecules present in a transformed

E.coli cell, but this number can be amplified by

incubating a log phase culture of CoIE1, carrying cells

in the presence of chloramphenicol. Like ColE 1,

pBR322 and all its derivatives are amplifiable, which

makes it very simple to isolate large amounts of these

plasmids. pBR322 contains two antibiotic-resistance

genes, one is the ampicillin-resistance (ampR) gene

coding for a β-Iactamase (which modifies ampicillin into

a form that is nontoxic to E. coli) and the other is the

tetracycline-resistance (tetR) gene (a set of genes

coding for enzymes that detoxify tetracycline)

Fig. 1a. pBR322 plasmid vector. (Source, Chawla, H.S.

2005. )

The complete nucleotide sequence of pBR322 has

been determined. The plasmid contains 20 unique

recognition sites for restriction enzymes. Six of these

sites, i.e. EcoRV, BamHI, Sphl, SalI, XmaIII and Nrul,

are located within the genes coding for tetracycline

resistance; two sites for HindIII and CIa I lie within the

promoter of the tetracycline resistance gene; and three

sites for PstI, PvuI and Sca l lie within the β-lactamase

gene. Cloning of a DNA fragment into any of these 11

sites results in the insertional inactivation of either one

of the antibiotic resistance markers. The gene of

interest is spliced into tetR gene cluster, and then the E.

coli cells are transformed. Thus, three types of cells are

obtained:

Cells that have not been transformed and so contain

no plasmid molecules and will be ampS tetS.

Cells that have been transformed with pBR322 but

without the inserted DNA fragment or gene will be

ampR tetR. These are transformed cells.

Cells that contain a recombinant DNA molecule, i.e,

the DNA fragment has been inserted into the

pBR322 at tetR gene cluster. These cells will lose

tetracycline resistance because the fragment has in-

serted in the middle of tetracycline resistance gene

cluster. These are recombinants and will be ampR

tetS.

Cloning foreign DNA in E. coli with pBR322.

If foreign DNA is inserted at the PstI restriction site, the

ampicillin resistance element is disrupted and inactivated.

After ligation of the DNA and transformation of E. coli

cells, the cells are grown on agar plates containing

tetracycline, to select for those that have taken up a

plasmid. Individual colonies from these agar plates are

transferred using sterile toothpicks to matching positions

within a grid on two additional plates; one plate contains

tetracycline (a control) and the other contains both

tetracycline and ampicillin. Those cells that grow in the

presence of tetracycline, but do not form colonies on the

plate containing tetracycline plus ampicillin, contain

recombinant plasmids (recall that the ampicillin-

resistance element becomes nonfunctional with the

insertion of foreign DNA). Cells that contain pBR322 that

was ligated without the insertion of a foreign DNA

fragment retain ampicillin resistance and grow on both

plates. Identification of recombinant clones requires

selection followed by screening. (Fig1b).

Fig.1b. Cloning of foreign DNA in E. coli with pBR322 (Source, David, L.N.

and Michael, M.C. 2001. )

pACYC184

This is a small 4.0 kb cloning vector developed from the

naturally occurring plasmid p15A (Fig 2). It contains the

genes for resistance to chloramphenicol and tetracycline.

The chloramphenicol gene has a unique EcoRI restriction

site and the tetracycline gene has a number of restriction

sites as mentioned in pBR322. Insertional inactivation of

one of the antibiotic resistance genes facilitates the

recognition of strains carrying plasmids with cloned DNA

fragments. Like pBR322, it is not self-transmissible, and

can be amplified. It has been a very popular cloning

vector since it is compatible with the ColE1-derived

plasmids of the pBR322 family and can, therefore, be

used in complementation studies.

Fig. 2. pACYC184 plasmid vector (Source, Chawla,

H.S. 2005. )

pUC vectors

The name pUC is derived because it was developed in the

University of California (UC) by Messings and his

colleagues (Norrander et al., 1983). These plasmids are

of 2.7 kb and possess the ColE 1 ori of replication. These

vectors contain ampR gene and a new gene called lacZ,

which was derived from the lac operon of E. coli that

codes for β-galactosidase enzyme (Fig.3).

Fig. 3. pUC plasmid vectors (Source, Chawla, H.S. 2005.

)

pUN121

This is a 4.4 kb vector derived from pBR322 (Nilsson et

al ,1983). This vector has been developed for rapid

selection of bacteria containing a recombinant plasmid.

This is required when genomic libraries are constructed,

which involves the construction of several thousands of

clones and handpicking of Individual clones with inserted

DNA would be extremely tedious. Direct selection vectors

have, therefore, been constructed. In these vectors (Fig.

4), the original promoter region of the tetracycline-

resistance gene is exchanged for the PR promoter of

bacteriophage (λ). Transcription from this promoter is

suppressed by the protein encoded by the lambda cl

gene, which was also cloned on pUN 121. Cells harboring

pUN 121 will, therefore, have the phenotype ampR tetS as

the cI gene product suppresses expression of the tetracy-

cline-resistance gene. The cl gene contains unique sites

for the restriction enzymes EcoRI, Hind II I, Ben, and

SmaI. Cloning of foreign DNA into these restriction sites

will cause insertional inactivation of the cl gene,

abolishing the suppression of PR promoter. Cells

harboring such a plasmid will become tetR. Plating of

transformed cells on plates containing tetracycline will

therefore only result in growth of cells harboring the

pUN121 vector with inserted DNA fragments.

a) b)

Fig. 4. pUN 121 plasmid vector(a positive selection vector)a-when no foreign

DNA is inserted in one of the restriction sites in the cl gene, the cl gene product

is synthesized and represses the P promoter. The cells harboring such plasmids

are sensitive to tetracycline, b- plasmids with foreign DNA inserted in the cl gene

produce a truncated repressor. The PL promoter is depressed and the cells

become tetracycline resistant. (Source, Chawla, H.S. 2005. )

Yeast plasmid vectors

Yeast (Saccharomyces cerevisiae) is a single celled

eukaryotic microbe, which can be cultured and

manipulated using the standard techniques applied to

bacteria. The genetic material of yeast is packaged into

chromosomes within a membrane-enclosed nucleus and

partitioned at cell division by mitosis and meiosis. In

1976, Struhl et al. reported that a fragment of yeast DNA

when cloned into E. coli restored histidine-independent

growth to strains carrying the hisB mutation. Similarly,

Ratzkin and Carbon (1977) ligated fragments of yeast

genome into plasmid CoIE1 and obtained a small number

of leucine independent transformants from a leuB E. coli

strain. In each case, the yeast chromosomal segment

carried the gene for the yeast enzyme equivalent to that

absent in the bacterial strain. Thus, the yeast HIS3 gene

was able to be expressed in a bacterial cell and produce

the yeast version of the enzyme imidazole glycerol

phosphate dehydratase and thereby complement the

mutation in the bacterial hisB strain. (With yeast genes it

is customary to indicate a wild type allele in capital

letters and the mutant allele in lower case letters.) Other

yeast genes isolated in this way include LEU2, URA3,

TRP1, and ARG4. In general, genes from eukaryotic

organisms rarely function in bacterial cells, but the

success with yeast genes arises from the lack of introns

in most such genes coupled with the ability of some

yeast promoters to function in bacteria.

The wild-type yeast genes thus isolated provided

essential markers in subsequent attempts to introduce

exogenous DNA into yeast cells. Hinnen et al. (1978)

demonstrated that the yeast LEU2 gene carried by the

plasmid Col E1 could be taken up by leu2 yeast

sphaeroplasts and transform them to a leucine-

independent phenotype. In this case the transformation

resulted from the integration of the added DNA into a

yeast chromosome. Thus, a variety of approaches have

resulted in a cloning system that makes use of many of

the powerful techniques developed for E. coli.

Episomal plasmids - Yeast episomal plasmid

(YEp). It is a yeast vector with the ability to replicate

autonomously without integration into a yeast

chromosome. Yeast vectors with this property have been

built around a naturally occurring yeast plasmid, the so-

called 2µ circle. This molecule has no function and hence

its name is based on contour length. It is present in most

yeast strains with a copy number of 40-50 per haploid

genome. The 2- µm plasmid, as it is called, is 6 kb in

size. These are autonomous and show high frequency

transformation.

Yeast integrating plasmids (YIps). These are not

actual yeast plasmid vectors, because they are unable to

replicate within a yeast cell without integration into a

chromosome. Such vectors contain a yeast marker as the

only addition to the bacterial plasmid. An important

development in the use of these vectors has been the

observation that opening the plasmid within the yeast

segment before adding it to the yeast cells greatly

increased the transformation frequency. For that

purpose, the plasmid is treated with a restriction enzyme

that has a unique recognition site within the yeast

segment. If a plasmid carried two yeast segments (e.g.,

URA3 and LEU2), cutting the plasmid within one of these

regions will target the integration to that chromosomal

locus.

Ti plasmids

Most cloning vectors for plants are based on the Ti

plasmid, which is not a natural plant plasmid, but belongs

to a soil bacterium Agrobacterium tumefaciens. This

bacterium invades plant tissues, causing a cancerous

growth called a crown gall. During infection, a part of the

Ti plasmid called the T-DNA is integrated into the plant

chromosomal DNA. There are Ri plasmids present in

Agrobacterium rhizogenes, which cause hairy root

disease in plants.

Organization of Ti plasmid

Ti plasmids of Agrobacterium are large circular DNA

molecules, up to 200 kb in length with molecular weights

of about 1.2 x 108 (3 to 8% of the Agrobacterium

chromosome). They exist in the bacterial cells as

independent replicating genetic units. Ti plasmids have

major regions for virulence, origin of replication,

conjugation, oncogenicity, and catabolism of opines.

Genetic organization of these regions has been shown in

octopine Ti plasmid DNA (Fig.5)

Fig. 5. Genetic map of an octopine Ti plasmid

(Source, Chawla, H.S. 2005. )

Vector systems

Most yeast vectors developed share the following

common features:

1. Most of· the vectors are derived from bacterial

plasmids and retain both the ability to replicate in

bacteria and selection markers suitable for use in

bacterial systems. As plasmid amplification is not high

in yeast as compared to that in E. coli, this is a more

useful feature. Thus, initial plasmid construction is

done in E. coli before transfer to yeast. Such vectors

permitting cloning in two different species are referred

to as shuttle vectors.

2. In most cases, the selection markers employed are

nutritional markers, such as LEU2 or HIS3. In some

cases, such markers are not suitable. For example,

Brewing yeasts are polyploid and it is, hence, not

possible to obtain auxotrophic mutants. In such cases,

resistances to copper or to the drug chloramphenicol

have been used.

Replication of Plasmids:

Most plasmid vectors in current use carry a replicon

derived from the plasmid pMB1, which was originally

isolated from a clinical specimen (its close relative is ColE

1 replicon). Under normal conditions of growth, 15-20

copies of plasmids carrying this replicon are maintained

in each bacterial cell. The pMB1 (or CoIE1) replicon does

not require plasmid-encoded functions for replication;

instead, it relies entirely on long-lived enzymes supplied

by the host. Replication occurs unidirectionally from a

specific origin and is primed by an RNA primer.

Replication of plasmid DNA is carried out by a subset of

enzymes used to replicate the bacterial chromosome.

Usually, a plasmid contains only one origin of replication

together with its associated cis-acting control elements.

The control of plasmid copy number resides in a region of

the plasmid DNA that includes the origin of DNA

replication.

The F plasmids encode the gene required for bacterial

conjugation. This extrachromosomal genetic element is

~95 kb, and was named because of its role in bacterial

fertility. The F plasmid is present as one copy per cell and

encodes ~100 genes, some of which are required for F

plasmid transfer during conjugation (tra genes). As a

result of integration into E. coli chromosome and then an

imprecise excision resulting in the reformation of a

circular plasmid, the F plasmid is able to transfer portions

of the E. coli genome into another bacterium via

conjugation. F plasmid containing E. coli strains are

required in molecular cloning involving M13 filamentous

bacteriophage. The F plasmid carries the sex pili gene,

which encodes the cell-surface binding site for M13

phages.

Some important features of cloning vectors

A plasmid vector used for cloning is specifically

developed by adding certain features:

Reduction in size of vector to a minimum to expand

the capacity of vector to clone large fragments. Since

the efficiency of transformation of bacterial cells

drops drastically when plasmids larger than 15 kb

are used, the size of cloning vector should be small,

preferably 3-4 kb. In this way, foreign DNA

fragments of 10-12 kb can be accommodated.

It should contain an origin of replication that

operates in the organism into which the cloned DNA

is to be introduced.

Introduction of selectable markers.

Introduction of synthetic cloning sites termed

polylinker, restriction site bank, or polycloning sites

that are recognized by restriction enzymes. This

polycloning site is usually present inside a marker

gene so that with the insertion of foreign DNA it will

inactivate that marker gene and the recombinants

can be selected.

Incorporation of axillary sequences, such as visual

identification of recombinant clones by histochemical

tests, generation of single stranded DNA templates

for DNA sequencing, transcription of foreign DNA

sequences in vitro, direct selection of recombinant

clones and expression of large amounts of foreign

proteins.