2. Plasmids
A plasmid is a small, extrachromosomal DNA
molecule within a cell that is physically separated
from chromosomal DNA and can replicate
independently.
They are most commonly found as small circular,
double-stranded DNA molecules in bacteria;
however, plasmids are sometimes present in archaea
and eukaryotic organisms.
3. Types of plasmids
Depending on their transmissibility property are of
three types:
1. Transmissible plasmid: They can be transferred
from cell to cell by the process of genetic transfer
like conjugation, hence also known as conjugative
plasmid.
Large plasmids with molecular weight of 40-100
million.
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2. Non – transmissible: These cannot be transferred
from cell to cell during conjugation as they lack
transferase gene.
They are small plasmids of molecular weight 3-20
million and are non-conjugative.
3. Episome: This plasmid lies either freely in circular
from or gets integrated into the host chromosomes.
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Depending on the nature of factors or function,
plasmids are of following types;
1. The F factor:
Also called fertility- factor or sex-factor.
It contains genetic information essentail for
controlling the matting process of bacteria during
conjugation.
These genes determine: Expression of pilli and
transfer of DNA during mating.
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2. The R factor: Also known as resistance factor.
Occur in 2 forms: Large plasmid and small plasmid.
Large plasmids are conjugative ‘R’ factors helps in
conjugation.
The small plasmids only contain ‘r’ genes and are not
conjugative.
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3. Colcinogenic (Col) factor:
Also known as bacteriocinogenic factor.
Col factor codes for the production of bacteriocins (
e.g; colicins, dipthericin, pyocyanin etc), which are
antibiotic like substances that are specifically and
selectively lethal to other closely related bacteria.
4. Virulence plasmid:
This plasmid codes for the virulence factor in some
bacteria that increases it pathogenicity.
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5. Metabolic plasmids:
This plasmid helps in various metabolic activities in
bacteria.
E.g : Root modulation and N2 fixation genes of
rhizobium are present in its plasmid.
9. Purification of plasmids
In biochemical aspects, to purify plasmid DNA from
bacteria is to isolate only plasmid DNA from the
mixture of biopolymers such as protein, ribonucleic
acid (RNA), chromosomal DNA and plasmid DNA,
by which bacteria cell is composed
10.
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Multiple methods of nucleic acid purification exist.
All work on the principle of generating conditions
where either only the nucleic acid precipitates, or
only other biomolecules precipitate, allowing the
nucleic acid to be separated.
Ethanol precipitation
Ethanol precipitation works by using ethanol as
an antisolvent of DNA, causing it to precipitate out
of solution. The soluble fraction is discarded to
remove other biomolecules.
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Spin column
Spin column-based nucleic acid
purification precipitates nucleic acid such that it binds
a solid matrix and other components flow through.
The conditions are then changed to elute the purified
nucleic acid.
Phenol–chloroform extraction
In a phenol–chloroform extraction, addition of
a phenol/chloroform mixture will dissolve protein
and lipid contaminants, leaving the nucleic acids in
the aqueous phase.
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It also denatures proteins, like DNase, which is
especially important if the plasmids are to be used
for enzyme digestion. Otherwise, smearing may
occur in enzyme restricted form of plasmid DNA.
14. Applications of plasmid
Plasmids are used in genetic engineering to amplify,
or produce many copies of certain genes.
They are used in different techniques and are
involved in research of genetic engineering and gene
therapy by gene transfer to bacterial cells or to cells
of superior organisms, whether other plants, animals
or other living organisms, to improve their resistance
to diseases, growth rates, or any other required traits.
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In molecular cloning, plasmids are types of vectors that are
useful in cloning short segments of DNA. For example, the
artificial and cost-effective bulk production of antibiotics can
be achieved by incorporating an expression vector for that
antibiotic in microbial cells. Similarly, other biomolecules can
also be produced.
In addition, plasmids are used to administer gene therapy,
which is a technique used to correct defective genes
responsible for disease development.
They can also be used to replicate proteins, such as the protein
that codes for insulin, in large amounts
16. Phage genetics
The most intensively studied bacteriophage is the
phage called lambda.
It is an important model system for the latent
infection of mammalian cells by retroviruses , and it
has been widely used for cloning purposes.
Lambda is the prototype of a group of phages that
are able to infect a cell and redirect the cell to
become a factory for the production of new virus
particles
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Lambda and other phages, which can establish lytic
or lysogenic cycles, are called temperate phages.
Other examples of temperate phages are
bacteriophage mu and P1.
Mu inserts randomly into the host chromosome
causing insertional mutations where intergrations
take place.
The P1 genome exists in the host cell as an
autonomous, self-replicating plasmid.
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Phage gene expression during the lytic and lysogenic
cycles uses the host RNA polymerase, as do other
viruses. However, lambda is unique in using a type of
regulation called antitermination.
19. Phage genetic organization
Phages are simple organisms that consist of a core of
genetic material (nucleic acid) surrounded by
a protein capsid.
The nucleic acid may be either DNA or RNA and
may be double-stranded or single-stranded. There are
three basic structural forms of phage: an icosahedral
(20-sided) head with a tail, an icosahedral head
without a tail, and a filamentous form.
20. Gene Mapping in plasmids
Gene mapping describes the methods used to
identify the locus of a gene and the distances between
genes.
The essence of all genome mapping is to place a
collection of molecular markers onto their respective
positions on the genome. Molecular markers come in
all forms. Genes can be viewed as one special type of
genetic markers in the construction of genome maps,
and mapped the same way as any other markers.
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22.
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The black arrows show the direction of transcription,
which is essential for cloning. If you clone your gene
of interest in a middle of another gene, make sure
that both of them are transcribed in the same
direction. Otherwise, the native promoter can
interfere with your gene expression.
plasmid sequences are diagrammed as linear
sequences starting from the ori. “Ori” means the
origin of plasmid replication.
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pBR322 has two antibiotic resistance
genes: tet (tetracycline resistance)
and amp (ampicillin resistance). These genes encode
an efflux pump (tetR) and beta-lactamase (ampR) to
excrete tetracycline and ampicillin from the cell,
respectively. Tet and amp are read in different
directions.
