Plasmids are small, circular, double- stranded, extrachromosomal DNAs present in bacterial

cells. They are inherited sharply with- out the influence of chromosomal DNA. They
replicate independently due to the presence of an origin of replication
The plasmids are 1 kbp- 200 kbp in size and have limited number of genes.
Most bacteria contain more than one copy of each plasmid. The number of copies of a
plasmid present in a cell is called copy number. The copy number of plasmids usually varies
from 1 to 50. However, it can be further increased by treating the bacterial culture with
chloramphenicol (an inhibitor of protein synthesis).
The genes for antibiotics resistance, nitro- gen fixation, nodulation, environmental stresses,
etc. occur in plasmid DNAs. The antibiotics resistance in plasmids can be used as genetic
marker to identify those strains which contain the plasmids. Some plasmids code for some
secondary metabolites.
Some plasmids, under certain conditions, integrate into the chromosomal DNA of the
bacterium. Such plasmids are called episomes. The integrated plasmid replicates along with
the chromosomal DNA. E.g., F-plasmid.
The eukaryotes, except yeasts, do not have plasmids. The yeast Saccharomyces cerevisiae
contains YEp (yeast episomal plasmid or 2-micron plasmid), YIp (yeast integrating plasmid)
and ARS (Automatically replicating sequence) in the cells.

Classification of Plasmids
On the basis of conjugative transfer, plasmids are classified into two categories. They are-
Conjugative Plasmids: These plasmids are transferred from one bacterium to the other
during conjugation. They contain 'tra genes for the conjugative transfer. E.g., F-plasmid.
Non-conjugative Plasmids: These plasmids do not pass from one bacterium to an- other
bacterium during conjugation. E.g., Col El plasmid.
On the basis of the functions, plasmids are classified into five types. They are-
F-Plasmid: F-plasmid or fertility plasmid contains some genes expressing the maleness in
bacteria. The genes are known as 'tra genes'. E.g., F-plasmid of E. coli.
R-Plasmids: R-plasmids (Resistance plasmids) contain some genes giving resistance to
bacteria against antibiotics and heavy metals. E.g., PSC101 gives the organism tetracycline
resistance
Col Plasmids: Col Plasmids code for the synthesis of bacterial toxin, colicin. Colicin kills
other closely related strains of the bacteria. E.g., Col El, Col B, etc.
Degradative Plasmids: Degradative plasmids code for enzymes that degrade toxic
substances such as toluene, xylene, salicylic acid, parathion, 2,4-D, etc. TOL plasmid of
Pseudomonas putida involves in the breakdown of toluene.
Virulence Plasmids: Virulence plasmids provide pathogenicity to bacteria.
Isolation and Purification of Plasmids
Characteristics of Ideal Plasmid Vectors
The ideal plasmid vectors must have the following characteristic features-
Size: The plasmid must be small in size. The small size is helpful for easy uptake of chimeric
DNA by host cells and for the isolation of plasmid without damage. The size of common
plasmid vectors ranges from 3kb to 8.5 kb. PRK646 plasmid is 3.4kb in size and pBR324 is
about 8.3kb in size.
Copy number: The plasmid must be present in multiple copies.
Genetic markers: The plasmid must have one or a few genetic markers. These markers help
us for the selection of organism that has recombinant DNA. Amp', Kan', Tet', Elimm, Ery',
Str and LacZ genes are used as genetic markers in plasmid vectors.
origin of Replication: The plasmid must have its own origin of replication and regulatory
genes for self-replication.
Unique Restriction Sites: The plasmid must have unique restriction sites for com- mon
restriction enzymes in use. Eg. EcoRI, Sacl, Kpnl, Xmal, Smal, BamHI, Xbal, HindIII.
BspMI, PstI and HindII.
Insertional Inactivation: The plasmid must have unique sites for restriction enzymes in
marker genes. This will help us for the selection of recombinants by insertional inactivation
method. For example, LacZ gene marker has unique sites for KpnI, Xmal and Bam HI.
Pathogenicity: The plasmid should not have any pathogenic property.
Introduction:
pBR322 is a purpose built plasmid vector and was one of the first widely used cloning vector.
It has a relatively small size of 4,363 bp. This is important because transformation efficiency
is inversely proportional to size and above 10 kbp is very low.
The nomenclature of PBR 322:
Created in 1977 in the laboratory of Herbert Boyer at the University of California, San
Francisco.
'p' indicates as a plasmid
'BR' identifies Bo-liver and Rodriguez, the two researchers who developed it
'322' distinguishes those plasmids from others (like PBR 325, PBR 327, etc.) developed in the
same laboratory.
pBR322 is 4361 base pairs in length and has two antibiotic resistance genes:
The gene bla encoding the ampicillin resistance (Amp) protein
The gene tet4 encoding the tetracycline resistance (Tet®) protein.
It contains the origin of replication of pMB1, and the rop gene, which encodes a restrictor of
plasmid copy number.
The plasmid has unique restriction sites for more than 40 restriction enzymes
11 of these 40 sites lie within the TetⓇ gene
There are two sites for restriction enzymes HindIII and Clal within the promoter of the Tet
gene.
There are six key restriction sites inside the AmpR gene.
Construction of pBR322:
pBR322 is NOT a naturally occurring plasmid.
It is manufactured by following steps:
The plasmid was constructed with genetic material from 3 main
The ampR gene originally resided on the plasmid RSF 2124 (a naturally occurring antibiotic
resistant plasmid in E. coli) The tet is derived from pSC101 (a second antibiotic resistant
plasmid)
The origin of replication is derived from pMB1, which is closely related to the colicin
producing plasmid ColE1

Recombinant selection with pBR322 is done by insertional inactivation of an antibiotic

resistance gene.
For example, A recombinant pBR322 molecule, one that carries an extra piece of DNA in the
BamHI site is no longer able to confer tetracycline resistance on its host, as one of the
necessary genes is now disrupted by the inserted DNA. Cells containing this recombinant
pBR322 molecule are still resistant to ampicillin, but sensitive to tetracycline (ampR tefs).
Uses of pBR322:
It is widely used as a cloning vector. In addition to this, it has been widely used as a model
system for study of prokaryotic transcription and translation.
Advantages of pBR322:
1. Small size (~ 4.4 kb) enables easy purifi-cation and manipulation.
2. Two selectable markers (amp and tet) al-low easy selection of recombinant DNA.
3. It can be amplified up to 1000-3000 copies per cell when protein synthesis is blocked by
the application of chloramphenicol.
Disadvantages of pBR322:
1. It has very high mobility i. e; it can move to another cell in the presence of a
conjugative plasmid like F-factor. Due to this, the vector may get lost in a population
of mixed host cells.
2. There is a limitation in the size of the gene of interest that it can accommodate.
3. Not a very high copy number is present as is expected from a good vector.
4. The screening process time-consuming and laborious.

pUC8
The pUC8 plasmid was designed by scientists and contains the lac z gene. To produce the
plasmid, the pBR322 plasmid was cut in half with EcoR I and the section containing the
ampicillin resistance gene was combined with a DNA fragment containing the lac z gene. As
a result, the plasmid provides a transformed cell with both ampicillin resistance and the
ability to utilize lactose as a food source, since the lac z gene produces B- galactosidase
(degrades lactose).

Since the pUC8 plasmid was produced using EcoR I, this enzyme cannot be used to insert
desired genes when producing recombinant plasmids for use in genetic engineering.
This plasmid was originally used in conjunction with a special mutant strain of E. coli called
JM101, which has a mutation in its chromosomal lac z gene and cannot survive on media
containing only lactose as a food source. If JM1010 bacteria were successfully transformed
with the pUC8 plasmid, they gained the ability to use lactose as a food source. Therefore,
only successfully transformed bacteria would survive when grown on media containing only
lactose and ampicillin.
Improving the use of pUC8
While it was possible to isolate the bacteria transformed with pUC8 on lactose and ampicillin
containing media, a problem existed. During the process of producing recombinant pUC8
plasmids with additional genes of interest inserted in them, some plasmids did not
successfully incorporate the desired gene and closed back up into non- recombinant plasmids.
When bacteria cells were exposed to the mixture of plasmids, the non-recombinant plasmids
were taken into cells along with the recombinant plasmids. Unfortunately, the non-
recombinant plasmids also gave cells the ability to metabolize lactose and survive in the
presence of ampicillin.

The solution to the problem of separating the JM101 cells transformed with recombinant
plasmids from the JM101 cells transformed with non-recombinant plasmids was solved using
the chemicals called X-Gal and IPTG. IPTG acts as an inducer that causes B- galactosidase to
bind to X-Gal. When X-Gal is broken down, it turns blue in color. Thus, all the JM101
bacteria that had the plasmid with an active lac z gene (producing B- Galactosidase) would
appear blue. The key to using this was to insert the additional desired gene in a location that
would disrupt the lac z gene in the plasmid and use media containing lactose, ampicillin, and
other nutrients. By doing so, all the bacteria with the recombinant plasmid would appear
white and all the bacteria with the non-recombinant plasmid would appear blue. The non-
transformed bacteria would be killed by the antibiotic and fail to grow. An additional benefit
was that scientists were not restricted to using E. coli from strain JM101. The technique could
be successfully employed with any type of bacteria cells that was used.
 Pgem3z
Similar to pUC Speialized for transcription as additional RNA pol binding site has been
added
RNA pol of phages can also be used; just change the recognition sequence as per requirement
and infect the bacterial cells with phage!!
It is very similar to pUC vector and is of same size (2750bp).
It carries the amp and Lac Z gene. The cluster of restriction sites is present in the Lac Z gene.
It has two promoter sequences i.e. T7 promoter (RNA polymerase of T7 bacteriophage) and
SP6 (RNA polymerase of SP6 phage) promoter sequences that lie on the either side of the
cluster of restriction sites.
These promoters act as the sites for the attachment of RNA polymerase. Thus if a
recombinant pGEM3Z is mixed with RNA polymerase in a test tube, transcription occurs and
RNA copies of the cloned fragment are synthesized.
Is pgem3z an expression vector?
The pGEM®-3Z Vector is intended for use as a standard cloning vector, as well as for the
highly efficient synthesis of RNA in vitro. The vector carries the lacZ α-peptide and the
multiple cloning region arrangement from pUC18 allowing selection of recombinants by
blue/white screening.
What is the copy number of pgem3z?
This plasmid contains a 147 bp nucleosome positioning sequence and is referred to as
clone"603"