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Construction of-WPS Office

The document discusses the construction of recombinant DNA and introduction of recombinant DNA into cells. It describes how foreign DNA fragments can be inserted into plasmid vectors, and then introduced into bacteria like E. coli through transformation, transduction, or conjugation to produce recombinant proteins.

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deepak gupta
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41 views11 pages

Construction of-WPS Office

The document discusses the construction of recombinant DNA and introduction of recombinant DNA into cells. It describes how foreign DNA fragments can be inserted into plasmid vectors, and then introduced into bacteria like E. coli through transformation, transduction, or conjugation to produce recombinant proteins.

Uploaded by

deepak gupta
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPTX, PDF, TXT or read online on Scribd
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Construction of recombinant

DNA & introduction of recombi


nant
DNA into cells

By Dr Deepak Rajpurohit
Construction of recombinant DNA, in which a
foreign DNA fragment is inserted into a plasmid
vector. In this example, the gene indicated by the
white color is inactivated upon insertion of the
foreign DNA fragment.
Recombinant DNA (rDNA) molecules are DNA
molecules formed by laboratory methods of genetic
recombination (such as molecular cloning) to bring
together genetic material from multiple sources,
creating sequences that would not otherwise be
found in biological organisms. Recombinant DNA is
possible because DNA molecules from all organisms
share the same chemical structure. They differ only
in the nucleotide sequence within that identical
overall structure.
Introduction

Till now, we have already discussed restriction enzymes


used for cutting/digesting of DNA fragments, vectors for
acting as carriers of DNA fragments and
insertion/ligation of DNA fragments into vector. The
next step in this process is to introduce recombinant
vectors carrying the cloned gene into appropriate host
including bacteria and animals which will be the subject
of this chapter.
Cloning in Bacteria

The most common model organism used as a potential host


for cloning is E. coli since it is very convenient to use.
Besides, a large number of E. coli vectors are available.
Moreover, the recombinant DNA can be easily introduced
into E. coli by transformation and a high level expression of
recombinant proteins can be obtained with commercially
available vectors. However, sometimes some important
functional genes need to be expressed in other organisms
like lactic acid bacteria including Lactobacillus spp.,
Pseudomonas spp., Bacillus spp. besides yeast and fungal
spp. In this chapter, we will confine to cloning in E. coli.
One of the pre-requisites is the need for a protocol to
introduce the recombinant DNA in bacteria and we will
concentrate on the methods used in E. coli since all the
prokaryotic or eukaryotic genes need to be sub cloned
into E. coli before they can be cloned and expressed in
other hosts.
Introduction of recombinant DNA in E. coli
The recombinant/vector DNA can be introduced in E. coli
using three gene transfer systems namely Transformation.
However, before we proceed to methods of introduction of
recombinant DNA into E. coli, we will discuss the gene
transfer systems operating naturally in bacteria in brief so
that the students can get adequate insight into these
systems operating in bacteria.
Transformation

Transformation is defined as the process of taking up of


naked DNA from the surroundings into the host cell.The
process of transformation was first demonstrated in
Streptococcus penumoniae by Frederick Griffith in 1928
who named the active principle as the transforming
principle.which converted rough cells into smooth cells in
the presence of heat killed smooth cells of S.
penumoniae leading to death of mice. The experiment
performed by Griffith .Later, Avery, MacCleod, McCarty in
1944 named this transforming principle as DNA.
Transformations can be brought about by the
following methods.

1. Transformation by Natural competence


2. Transformation by Induced competence
3. Protoplast Transformation and Protoplast fusion
4.Electroporation
Transduction
Transduction is the process of transfer of bacterial genes
into a host bacterium with the help/aid of phages.
Transduction is also known as phage mediated genetic
transfer (Fig. 12.1). Transduction is of two types:
a) Generalized transduction
When a phage particle attacks a bacterial cell and during
its assembly, fragments of host DNA are packaged into
phage particles in place of its own DNA. When such a
phage/virus particle infects a new host cell, DNA is
injected into the host which can recombine with the host
genome (Fig. 12.3).
b) Specialized transduction

A lysogenic phage undergoes recombination with


the host genome at a particular site e.g. gal and bio
as shown in Fig. 12.4a and later when it is excised to
become an independent phage genome, it carries
one or more host genes with it (Fig. 12.4b). These
genes can be transduced into a new host cell which
recombine into the genome of that cell.
Conjugation
Conjugation is defined as the process of transfer of genetic
material from a donor cell to a recipient cell through a
mating bridge . It was discovered by Lederberg and Tatum
in 1946 who showed that two different strains of bacteria
with different growth requirements could exchange
genes . They surmised that the bacterial cells must
interact with each other in order to transfer the genetic
material and the process is now known as sexual
conjugation by direct contact. A segment (rarely all) of the
donor’s chromosome recombines with the homologous
recipient chromosome. Recipients containing donor DNA
are called transconjugants. Bernard Davis demonstrated
that the physical contact is essential for transfer of genetic
material.

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