BIOCHEMISTRY

B. Sc.
3rd Semester

PAPER NO. : BCH 303

Paper: Microbiology

TOPIC NO. 6
Bacterial genetics

Lecture -195
BACTERIAL GENETICS II
(TRANSFORMATION, TRANSDUCTION AND TRANSFECTION)

INTRODUCTION

Recombination is a process in which a new recombinant chromosome, one with a

genotype different from either parent is formed by combining genetic material from two
organisms. Recombination results in a new arrangement of genes or parts of genes and is
normally accompanied by a phenotypic change. Microorganisms can carry out different
types of recombination. There are very effective three modes of gene transfer seen in
bacteria which includes the transformation, transduction and transfection. This methods
of genes transfer leads to the development of molecular cloning. The basic principle of
molecular cloning is the transfer of desired gene from donor to a selected recipient for
various applications in the field of medicine, research, gene therapy with an ultimate aim
of beneficial to the mankind.

TRANSFORMATION

Transformation is the uptake of a naked DNA molecule or fragment by a cell from the
medium and incorporate into the recipient chromosome in a heritable form. The process
of transformation was discovered by Fred Griffith in the year 1928. At the time of the
lysis of the bacterial cell, it leads to the release of large amounts of DNA into the
surrounding environment. These DNA fragments may contain several genes and if this
bacterial DNA fragments comes in contact with a competent cell i.e. one that is able to
take up the DNA, and be transformed, it can bound to the cell and the DNA can be taken
inside. The frequency of transformation of a competent cell is approximately 10-3 when an
excess of the DNA is used. Competency is a complex phenomenon and is dependent on
several conditions of the bacterial growth. Here, taking an example of Streptococcus
pneumoniae, they become competent during the exponential growth phase i.e. when the
bacteria is actively dividing or when the population reaches about 107 to 108 cells per ml.
when the population becomes competent the bacteria secretes a protein known as the
competence factor which function to stimulate the production of 8 to 10 new proteins
required for transformation.

Artificial transformation can also be carried out in the laboratory by treatment of the cells
with calcium chloride. This renders the membrane more permeable to DNA. Relatively
high concentrations of DNA that is higher than would normally present in nature are used
to increase the transformation frequency. It is easier to transform bacteria with plasmid
DNA since plasmids are not easily degraded as linear fragments and can replicate within
the host cell.
TRANSDUCTION

Transduction is the process by which DNA is transferred from one bacterium to another
by a virus . Transduction does not require physical contact between the cell donating the
DNA and the cell receiving the DNA. Transduction was first describe by Joshua Lederberg
and Norton Zinder in 1952. There are two types of transduction –

1. Generalized transduction
2. Specialized transduction
GENERALIZED TRANSDUCTION

Generalized transduction occurs during the lytic cycle of the virulent or temperate
phages. During assembly when the viral chromosomes are packaged into the capsid,
random fragments of the partially degraded bacterial chromosomes also may be packaged
by mistake. As the capacity of the capsid is limited in quantity of the DNA to be packed,
some viral DNA is left behind. Hence, the quantity of bacterial DNA carried depends on the
size of the capsid. The resulting virus particle infects another bacterial cell but does not
initiate lysis. Therefore, this phage is known as a generalized transducing particle.
Generalized transduction can be either complete or abortive. In complete transduction
the transduced DNA fragments gets integrated into the recipient bacterial chromosome
forming a recombinant chromosome and this DNA fragment replicate along with the
bacterial chromosome replication and passed on to the daughter cells. While in the
abortive transduction the transduced DNA fragment may not integrated into the bacterial
chromosome and remains in the cytoplasm as free particles and they cannot undergo
replication.
SPECIALIZED TRANSDUCTION

Specialized transduction is also known as the restricted transduction. In this type of

transduction, the transducing particle carries only specific portions of the bacterial
genome. Specialized transduction occurs when there is an error in the lysogenic cycle of
the phage. When a prophage is induced to leave the host chromosome, excision
sometimes may be carried out improperly and hence the resulting phage genome
contains a portion of the bacterial chromosome which is about 5 – 10 % of the bacterial
DNA located just next to the integration site. Here, a transducing particle injects the
bacterial genes into another bacterium. One of the best studied example of specialized
transduction is the lambda phage.
TRANSFECTION

Transfection is the process of introducing foreign molecules and genetic materials into
eukaryotic cell. It is one of the method of gene transfer where the genetic material is
introduced into the animal cell with an aim to study various functions of proteins and the
gene. The term transfection is commonly used for non-viral methods of gene delivery in
eukaryotic cells. Transfection is commonly used in biological laboratories for studying,
modulation of gene expression, gene function, gene regulation, protein expression,
biochemical mapping, mutational analysis and protein production.

This mode of gene transfer involves creation of pores on the cell membrane enabling the
cell to receive the foreign genetic material. The significance of creating pores and
introducing the DNA into the host mammalian cell contributed to different methods in
transfection. Chemical mediated transfection involves use of either calcium phosphate or
cationic polymers or liposomes. Electroporation, sonoporation, optical transfection, hydro
dynamic delivery are some of the non-chemical based gene transfer. Particle based
transfection uses gene gun technique where a nanoparticle is used to transfer the DNA to
host cell.

There are two main types of transfection –

 1. transient transfection and
2. stable transfection.
Transient transfection is the transfection in which the foreign DNA is eliminated by the
cell prior to or during mitosis. Thus the foreign DNA does not carry over to the new cell
and they are unaffected. Whereas stable transfection is the form of transfection in which
the foreign DNA becomes parts of the cell’s original DNA by either adding on to it or
replacing the old DNA to create new DNA strand. Transfection methods can be divided
into three groups –
1. Methods that rely on carrier molecules
2. Methods that deliver nucleic acids directly to the cytoplasm.
3. Methods that employ viral vectors

Transfection using calcium phosphate was one of the earliest method developed to
introduce foreign molecule to a cultured mammalian cell. In this method, calcium chloride
containing HEPES-buffered saline solution is used to deliver plasmid DNA. Development
of lipid and polymer based carrier molecules having capacity to bind nucleic acids led to
the adaptation of these compounds in transfection. Liposomes formed from these
compounds are able to fuse with cellular membranes by which they can deliver bound
DNA or RNA to the new cell. The transfection reaction is commonly performed under
aqueous conditions. Some of the problems associated with traditional transfection
methods like calcium phosphate co-precipitation, DEAE- dextran, polybrene and
electroporation, include low efficiency of DNA delivery, poor reproducibility, cell toxicity
and inconvenience. The cationic lipid reagent- mediated transfection yields high and
previously unattainable transfection effiencies in a wide variety of eukaryotic cells.
With cationic lipid reagents, the DNA solution is not deliberately encapsulated within the
liposomes, rather, the negatively charged DNA binds spontaneously to the positively
charged liposomes, forming DNA–cationic lipid reagent complexes.

Transfection of adherent cells using various agents such as lipofectamine requires

optimization of amount of plasmid or reagent and cell number and specific for cell type.
These molecules can work in a number of different ways to carry DNA into the target cells
and insert it into their genome. The typical process involves mixing the desired DNA with
the reagent, adding it to cells in culture, incubating them, and then identify them, which
contain the new DNA. The use of viral DNA vectors as well as mechanical methods like
microinjection, where the desired DNA is directly injected to reduce the risk of degrading
the material. Gene guns are another popular tool for DNA transfection.

CONCLUSION

Bacterial recombination is a one-way process in which the fragment of DNA is transferred

from the donor to a recipient and is integrated. There are very effective three modes of
gene transfer seen in bacteria which includes the transformation, transduction and
transfection. Transduction is the transfer of bacterial genes by the viruses and
transformation is the uptake of the naked DNA molecule by a competent cell and is
incorporated into the genome while transfection is the process of introducing foreign
molecules and genetic materials into eukaryotic cell. Finally, the transfer of desired gene
from donor to a selected recipient has a wide application in the field of medicine,
research, gene therapy which aimed to benefit the human mankind.