HEAT SHOCK AND

CHEMICAL TREATMENT
NAME-KAPIL SINGH
ROLL NO.-30
Biotechnology consists of a major field of research known as Genetic engineering,
which is the process of using recombinant DNA (rDNA) technology to alter the
genetic makeup of an organism.

Recombinant DNA technology is the joining together of DNA molecules from two
different species.

This process of genetic engineering done in multiple steps including-

• identification of the section of DNA that contains the required gene from
source chromosome,

• extraction of the required gene,

• insertion of the required gene into a bacterial plasmid or vector,

• insertion of that plasmid into host cell

• and growing the transformed cells to produce a Genetically Modified

organism.
 
After the first 3 steps are completed that is selection of the gene of interest, its extraction
and the isolation of that gene by the help of a bacterial plasmid, the plasmid has to be
inserted into the host cell.

Bacteria is used as the host cell for accepting the recombinant plasmid.
The plasmid used in this process has both the required gene and one other gene that
makes it resistant to a particular antibiotic that is used further in the process.

The processes in whole is termed as transformation of bacteria which 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.

There are many methods to do this and one of those methods is using heat shock.
TRANSFORMATION OF BACTERIA

 Transformation is the process that occurs when a cell ingests foreign DNA
from its surroundings. Transformation can occur in nature in certain types of
bacteria.
 In molecular biology, transformation is artificially reproduced in the lab via
the creation of pores in bacterial cell membranes.
 Bacterial cells that are able to take up DNA from the environment are called
competent cells. In the laboratory, bacterial cells can be made competent and
DNA subsequently introduced by chemical treatment and heat shock.
 PROCESS OF HEAT SHOCK

 Chemically competent cells from host cell are created using a series of cold salt
washes to disrupt the cell membranes, preparing the cells to accept plasmid
DNA.
 After this the plasmid DNA is added to the cells.
 Incubation of the competent cell/DNA mixture is done on ice for 20-30 mins.
 All of this is done in a calcium rich environment and the process is known as
chemical treatment which is done to bring the plasmid and host cell closer.
 To introduce the desired plasmid into chemically competent cells. The plasmid-
cell mixture then is put directly into a water bath having temperatures of 45–
50°C for 45 sec to 1 min.
 This extreme change in temperature opens up pores in the cell membrane of the
host cell which allows the plasmid to enter into the host cell.
CHEMICAL TREATMENT

 To make the bacterial cells capable to take up the plasmid from their surrounding, we
must induce competence in the cells.
 The cells are chemically prepared to be competent by calcium chloride treatment in
which they are first placed in a calcium rich environment.
 The CaCl2 serves two functions:-
1. To bring the plasmid closer to the host cell.
2.  To counteract the electrostatic repulsion between the plasmid DNA and
bacterial cellular membrane as both DNA and the cell membrane of the
bacteria is negatively charged. So the dual positive charge on the calcium
ions will help neutralize them.
 BACTERIA AND PLASMID WHEN PLACED
INSIDE THE WATERBATH
Pores begin to form in the The plasmid enters the host
bacterial cell membrane. bacteria through the pore.
 The heated mixture is then placed back on ice to retain the plasmids inside the bacteria and to
recover.

 Many cells do not survive the rapid temperature change but enough maintain integrity to
keep the plasmid.

 After this growth medium is added into the mixture and it is left to incubate at 37 C for some
time so that the cell membrane is able to regenerate and the cells are also able to divide to
some extent.

 The cells then attained are known as recombinant bacteria.

 The cells are then plated on a selective medium which contains antibiotics. The antibiotics
stop the growth of the bacteria that are not resistant to it. The plasmid that we had used had
the antibiotic resistant gene along with the gene of interest and is able to survive in the
medium it is placed in.
THANK YOU