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Bacterial transformation
1. NAME – SAMADRITA BANIK
ST. GEORGE COLLEGE OF MANAGEMENT
AND SCIENCE
M.Sc MICROBIOLOGY
2nd SEMESTER
2.
Bacterial transformation is a process of horizontal gene
transfer by which some bacteria take up foreign genetic
material (naked DNA) from the environment. It was first
reported in Streptococcus pneumoniae by Griffith in 1928.
DNA as the transforming principle was demonstrated by
Avery et al in 1944.
INTRODUCTION
3.
Bacteria can take up foreign DNA in a process called
transformation.
Transformation is a key step in DNA cloning. It occurs after
restriction digest and ligation and transfers newly made
plasmids to bacteria.
After transformation, bacteria are selected on antibiotic plates.
Bacteria with a plasmid are antibiotic-resistant, and each one
will form a colony.
Colonies with the right plasmid can be grown to make large
cultures of identical bacteria, which are used to produce
plasmid or make protein.
BACTERIAL
TRANSFORMATION
4.
Transformation and selection of bacteria are key steps in DNA cloning. DNA cloning is the
process of making many copies of a specific piece of DNA, such as a gene.
The copies are often made in bacteria.In a typical cloning experiment, researchers first insert
a piece of DNA, such as a gene, into a circular piece of DNA called a plasmid.
This step uses restriction enzymes and DNA ligase and is called a ligation.After a ligation,
the next step is to transfer the DNA into bacteria in a process called transformation.
Then, we can use antibiotic selection and DNA analysis methods to identify bacteria that
contain the plasmid we’re looking for.
DNA CLONING
6.
Specially prepared bacteria are mixed with DNA (e.g.,
from a ligation).
The bacteria are given a heat shock, which causes some of
them to take up a plasmid.
Plasmids used in cloning contain an antibiotic resistance
gene. Thus, all of the bacteria are placed on an antibiotic
plate to select for ones that took up a plasmid.
8. Bacteria without a plasmid die. Each bacterium with a plasmid
gives rise to a cluster of identical, plasmid-containing bacteria
called a colony.
Several colonies are checked to identify one with the right
plasmid (e.g., by PCR or restriction digest).
A colony containing the right plasmid is grown in bulk and
used for plasmid or protein production.
9.
Possibility 1: Bacteria = plasmid factories
In some cases, bacteria are simply used as "plasmid factories," making lots of
plasmid DNA. The plasmid DNA might be used in further DNA cloning steps
(e.g., to build more complex plasmids) or in various types of experiments.
In some cases, plasmids are directly used for practical purposes. For instance,
plasmids were used to deliver a human gene to lung tissue in a recent gene
therapy clinical trial for patients with the genetic disorder cystic fibrosis
Protein production in bacteria
10.
Possibility 2: Bacteria = protein factories
In other cases, bacteria may be used as protein factories. If a plasmid contains
the right control sequences, bacteria can be induced to express the gene it
contains when a chemical signal is added. Expression of the gene leads to
production of mRNA, which is translated into protein. The bacteria can then be
lysed (split open) to release the protein.
11.
Bacteria contain many proteins and macromolecules. Because of
this, the newly made protein needs to be purified before it can be
used. There are a variety of different techniques used for protein
purification.
12. In one technique called affinity chromatography, a mixture of molecules
extracted from the lysed bacteria is poured through a column, or a cylinder
packed with beads. The beads are coated with an antibody, an immune
system protein that binds specifically to a target molecule.
The antibody in the column is designed to bind to our protein of interest, and
not to any other molecules in the mixture. Thus, the protein of interest is
trapped in the column, while the other molecules are washed away. In the
final step, the protein of interest is released from the column and collected
for use.
13.
Transformation is the process by which foreign DNA is introduced
into a cell. Transformation of bacteria with plasmids is important
not only for studies in bacteria but also because bacteria are used as
the means for both storing and replicating plasmids. Because of
this, nearly all plasmids (even those designed for mammalian cell
expression) carry both a bacterial origin of replication and an
antibiotic resistance gene for use as a selectable marker in bacteria.
CONCLUSION
