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0 Introduction
Virtually all microbial traits are controlled or influenced by heredity. The inherited traits of microbes
include their shape and structural features, their metabolism. their ability to move or behave in various
ways, and their ability to interact with other organisms-perhaps causing disease. Individual organisms
transmit these characteristics to their offspring through genes
In 1866, the Czech-Austrian monk Gregor Mendel determined that traits are inherited as physical units,
now called genes. The precise function of genes, however, was not revealed until 1941, when George
Beadle and Edward Tatum published a scientific paper reporting that genes direct the production of
enzymes. Microorganisms, especially bacteria, have significant advantages as model organisms, in part
because of their unique characteristics.. The early work of Fred Griffith in 1928 on the transfer of
virulence (that is, the ability to cause disease) in the pathogen Streptococcus pneumoniae, commonly
called pneumococcus, set the stage for research showing that DNA was indeed the genetic material.
Oswald Avery and his colleagues then set out to discover which constituent in the heat-killed virulent
pneumococci was responsible for transformation.
Eight years later, Alfred Hershey and Martha Chase wanted to know if protein or DNA carried the
genetic information of a bacterial virus called T2 bacteriophage.
Codon A series of three nucleotides that code for a specific amino acid.
Gene The functional unit of the genome; it encodes a product, most often a
protein.
The genotype of an organism is its genetic makeup, the information that codes
for all the particular characteristics of the organism. The genotype represents
potelltial properties, but not the properties themselves.
Ribosome Structure that facilitates the joining of amino acids during translation;
composed of ribosomal RNA (rRNA) and protein.
RNA Polymerase Enzyme that synthesizes RNA using one strand of DNA as a
template.
Transcription The process by which the information encoded in DNA is copied into
RNA.
Transfer RNA (tRNA) Type of RNA molecule involved in interpreting the genetic
code; each tRNA molecule carries a specific amino acid.
Characteristics of RNA
DNA replication
DNA replication is very important in gene expression
Helicase
Single strand binding protein (SSB)
Primase
DNA polymerase
Exonuclease
DNA ligase
Helicase unwinds the DNA at a specific site called replication fork
Single strand binding protein stabilize the two separate strand in other to stop
them from annealing( joining back together)
Primase add a short segment of RNA called a primer so that DNA polymerase can
begin addition of nucleotide
Note; the directionality of replication is read up and write down from 3 to 5 prime
Single strand binding proteins (SSB) will be displace and move away. The new
strand will be produce in the 5 prime to 3 prime direction. And this process is
continues only in the leading strand
In the lagging strand DNA polymerase is unable to add up bases from 5 prime to 3
prime. To do that they need a new primer and a new DNA polymerase that will
add a short fragment of DNA called okazaki fragments. After adding a primer and
okazaki fragments an enzyme called exonuclease will eliminate the primer and
DNA polymerase will continue to add bases to the existing DNA. DNA polymerase
is incapable of joining with an already existing strand of newly synthesize DNA.
DNA ligase in an ATP dependent manner joins the two short DNA fragments or
components together, Note; the synthesis of two doughter DNA is semi
conservative.
Transcription
Transcription begins when RNA polymerase recognizes and binds to a promoter
region on the double-stranded DNA molecule.
Sigma factor binds to RNA polymerase and RNA polymerase binds to promoter to
form a closed promoter complex
It drags itself to a region, RNA polymerase encounters a terminator, it falls off the
DNA template and releases the newly synthesized RNA.
Translation
The first modified a. acid called F-met carried by tRNA will be attached to P-site.
In elongation, the second a. acid will be carried by the tRNA and attached it to the
A- site. When the second a. acid is in the A-site, ribosome will move along one
codon unit. The a. acid ( F-met) in p-site will form peptidyl bond with the amino
acid in A-site by an enzymes called peptidyl transferase
The tRNA that carries a. acid and an anticodon will bind with codon in mRNA.
Translocation of a. acid by the elongation factor EFGU will continue to occur from
A to P and P to E-site which will be exited.
The process of translocating the synthesis of the polypeptide chain will continue
until it reaches the stop codon UAA, UAA and UAG.
When they reaches the stop codon, all the structures ie (30s 50s), translation
factors and the tRNA will be dissociated for another synthesis to begin.
2.0 Bacterial Gene Regulation
In bacterial cells, many genes are routinely expressed, but others are
regulated in response to environmental conditions. A regulatory
mechanism sometimes controls the transcription of only a limited
number of genes, but in other cases, a wide array of genes is controlled
coordinately. A set of regulated genes transcribed as a single mRNA
molecule, along with the sequences that control its expression, is called
an operon. One of the most well-characterized examples is the lac
operon, which encodes proteins required for transporting and
hydrolyzing the disaccharide lactose.
Activators
Lactose and the lac Operon The lac operon uses a repressor that
prevents transcription when lactose is not available; the repressor
binds the operator, blocking RNA polymerase. When lactose is in the
cell, however, some of it is converted to allolactose, an inducer. This
compound binds the repressor and, in doing so, changes the repressor’s
shape so that it can no longer bind to the operator. With the operator
unoccupied, RNA polymerase can begin transcribing the operon.
However, this can happen only if glucose is not available in the growth
medium.
The target DNA or the DNA insert which has been extracted and cleaved
enzymatically by the selective restriction endonuclease enzymes [in step (i)] are
now ligated (joined) by the enzyme ligase to vector DNA to form a rec DNA
molecule which is often called as cloning-vector-insert DNA construct.
Suitable host cells are selected and the rec DNA molecule so formed [in step (iii)]
is introduced into these host cells. This process of entry of rec DNA into the host
cell is called transformation. Usually selected hosts are bacterial cells like E. coli,
however yeast, fungi may also be utilized.
Transformed cells (or recombinant cells) are those host cells which have taken up
the recDNA molecule. In this step the transformed cells are separated from the
non-transformed cells by using various methods making use of marker genes.
Exonuclease
DNA ligase:
The function of these enzymes is to join two fragments of DNA by synthesizing the
phosphodiester bond. They function to repair the single stranded nicks in DNA
double helix and in rec DNA technology they are employed for sealing the nicks
between adjacent nucleotides.
DNA polymerases
These are the enzymes which synthesize a new complementary DNA strand of an
existing DNA or RNA template. A few important types of DNA polymerases are
used routinely in genetic engineering. One such enzyme is DNA polymerase
Cloning vector
It is another important natural tool which geneticists use in rec DNA technology.
The cloning vector is the DNA molecule capable of replication in a host organism,
into which the target DNA is introduced producing the rec DNA molecule.Different
types of DNA molecules may be used as cloning vehicles such as plasmids,
bacteriophages, cosmids.
Host organism
A good host organism is an essential tool tor genetic engineering. Most widely
used host for rec DNA technology is the bacterium E. coli. because cloning and
isolation of DNA inserts is very easy in this host. A good host organism is the one
which is easy to transform and in which the replication of rec DNA is easier. There
should not be any interfering element against the replication of rec DNA in the
host cells
Linkers and adaptors are the DNA molecules which help in the
modifications of cut ends of DNA fragments. These can be joined to the
cut ends and hence produce modifications as desired.
Linkers contain target sites for the action of one or more restriction
enzymes. They can be ligated to the blunt ends of foreign DNA or
vector DNA.
Gel Electrophoresis
It is the technique of separation of charged molecules (in aqueous
phase) under the influence of an electrical field so that they move on
the gel towards the electrode of opposite charge i.e., cations move
towards the negative electrode and anions move towards the positive
electrode.
Blotting Techniques
Visualization of a specific DNA (or RNA or protein) fragment out of
many molecules requires a technique called blot transfer. In this
technique, the separated bands are transferred onto a nitrocellulose
membrane from the gel. Mainly there are three types of blot transfer
procedures:
Southern Blotting, Northern Blotting and Western blotting
DNA Sequencing
The segments of specific DNA molecules obtained by recombinant DNA
technology can be analysed for determining their nucleotide sequence.
The methods commonly used for DNA sequencing are:
The next step is the primer annealing. In this step the two primers, the
forward primers and the backward primers, anneal or hybridize to the
single-stranded template DNA at its complementary regions. Annealing
is usually carried out at a lower temperature depending on the length
and sequence of the primers. In standard cases it is in the range of 40
to 50°C.
The final step in each cycle is the extension in which the Taq DNA
polymerase synthesizes the DNA region between the primers using
dNTPs and Mg2+. The optimum temperature for carrying out the
primer extension reaction or polymerization of dNTPs is standardized at
72°C.
Main Requirements of PCR
Two nucleotide primers which are complementary to 3′ ends of
Uses of PCR:
Production of Hormones:
By the advent of techniques of rec DNA technology, bacterial cells like
E.coli are utilized for the production of different fine chemicals like
insulin, somatostatin, somatotropin and p-endorphin. The genes of
interest are incorporated into the bacterial cells which are then cloned.
Such clones are capable of producing a fair amount of hormones like
insulin which have great commercial importance.
Biosynthesis of Interferon
By recDNA technolology method, the gene of human fibroblasts (which
produce interferon’s in human beings) is inserted into the bacterial
plasmid.
Production of vaccines
A number of vaccines have been synthesized biologically through rec
DNA technology. DNA-vaccine is the preparation that contains a
gene encoding an immunogenic protein from the concerned
pathogen.
Production of Antibiotics:
recDNA technology helps in increasing the production of antibiotics by
improving the microbial strains through modification of genetic
characteristics.
Gene therapy
Gene therapy is undoubtedly the most beneficial area of genetic
engineering for human beings. It involves delivery of specific genes into
human body to correct the diseases. Thus it is the treatment of diseases
by transfer and expression of a gene into the patients’ cells so as to
ensure the restoration of a normal cellular activity.
On the basis of types of cells into which the functional genes are
introduced, the gene therapy may be classified as somatic gene therapy
and germ line gene therapy. Gene therapy is done either by using in
vivo strategy (also called as patient therapy) or by using the ex vivo
strategy.
Industrial application
Application in Enzyme Engineering:
As we know that the enzymes are encoded by genes, so if there are
changes in a gene then definitely the enzyme structure also changes.
Enzyme engineering utilizes the same fact and can be explained as the
modification of an enzyme structure by inducing alterations in the
genes which encode for that particular enzyme.
Biofuel Production
Biofuels are derived from biomass and these are renewable and cost
effective. Genetic engineering plays an essentially important role in a
beneficial and large scale production of biofuels like biogas. bio
hydrogen biodiesel bio-ethanol., etc. Genetic engineering helps to
improve organisms for obtaining higher product yields and product
Application In agriculture
Transgenic Plants:
A genetically modified plant, consisting in its genome, one or more
inserted genes of an unrelated plant is termed as a transgenic plant and
those inserted genes are called as transgenic. The development of
transgenic plant is possible by using recDNA technology, gene delivery
strategies and the tissue culture techniques.
This can be done by following any of the gene delivery systems available
like AMGT (Agrobacterium mediated gene transfer), using plant viruses
as vectors or by direct gene delivery system i.e., electroporation,
microinjection, particle-gun method, etc
Application in research
recDNA technology has an immense scope in Research and
Application in environment
Environment Protection
Genetic engineering makes its contributions to the environment
protection in various ways. Major approach in environment protection
is the use of recDNA technology for degradation of toxic pollutants
which harm the environment. Different microbes used for sewage
treatment, waste water treatment, industrial effluent treatment and for
bioremediation are greatly improved by genetic engineering practices
and thus present better results.