Strain improvement by genetic engineering

1. Genetic Recombination or Gene transfer.:
By
Dr. Harinatha Reddy A
ASSISTANT PROFESSOR
Department of Biotechnology
Bangalore

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3.  The strain improvement can be made by combining genetic
information from two genotypes, by a process called genetic
recombination.
The genetic recombination can be brought out by:
 Protoplast fusion
 Transformation,
 Transduction,
 Conjugation
 Genetic engineering methods.

4. Protoplast fusion:
 Protoplasts are naked cells without cell wall but they posses plasma
membrane and other cell components.
 Protoplasts of different species can be fused to generate a hybrid cell
this process is called protoplast fusion or somatic hybridization.
 The hybrid protoplast contain= cytoplasm+ 2 nucleus.

5. Enzymes for the preparation of protoplasts:
Plant cells: Cellulase, pectinase, xylanase.
Bacteria: Lysozyme (+EDTA).
Fungal cells: Chitinase.

6. Methods of Protoplast Fusion:

7. Spontaneous Fusion:
 Protoplasts, during isolation, often fuse spontaneously and this
phenomenon is called spontaneous fusion.
 Simple physical contact between two protoplasts to bring the
spontaneous fusion.

8. Chemo-Fusion:
 Polyethylene glycol (PEG), Calcium ions are the most commonly
used chemicals for protoplast fusion.
 Polyethylene glycol (PEG), Calcium ions induce agglutination of
protoplasts.

9.  High concentration of PEG and Ca2+ ions neutralise surface
charges on the protoplasts and allow contact of plasma membrane
of agglutinated protoplasts.

10. Electrofusion:

11.  In this method electrical field is used to for protoplast fusion.
 When protoplasts are placed in a culture vessel fitted with
microelectrodes and electric shock is applied protoplasts are induce
to fuse.
 This electro fusion technique is simple and quick.

12.  A low–voltage (AC) field is applied which aligns protoplasts into
chain of cells (pearl chains) between the two electrodes.
 Once alignment is complete, the fusion is induced by application of
a of high-voltage (DC ) pulses.

13.  A high voltage DC pulse induces breakdown of the plasma
membrane at the site of cell contact, leading to fusion and
reorganization of membrane.
 Entire process takes less than 5 minutes.

14. Transformation:

15.  In molecular biology, transformation is the genetic alteration of a
cell, resulting from the direct uptake and incorporation of
exogenous genetic material from its surroundings through the cell
membrane(s).
Recipient bacteria
Donor bacteria
Recombinant bacteria

16. Calcium chloride Method:
Bacterial cells incubated
With CaCl2 and DNA
at cold Temperature (4°C )
and followed by Heat shock
(45°C ).

17. Calcium chloride Method:
 Typically the bacterial cells are incubated in a solution containing
divalent cations (often calcium chloride) and DNA under cold
conditions, before being exposed to a heat pulse (heat shock).
 Calcium chloride partially disrupts the cell wall and membrane,
which allows the recombinant DNA enter the host cell.

18. Electroporation

19. Electroporation:
 In this method the bacterial cells are briefly shocked with an
electric field of 10-20 kV/cm.
 Which is thought to create holes in the cell membrane through
which the plasmid DNA may enter.
 After the electric shock, the holes are rapidly closed by the Cell
membrane-repair mechanisms.

20. Conjugation:

21. Conjugation:

22.  Bacterial conjugation is the transfer of genetic material between
bacterial cells through conjugation tube.
 It is unidirectional transfer of genetic material from one strain to
the other.
 The genetic information transferred is often beneficial to the
recipient.
 It include antibiotic resistance, xenobiotic tolerance or the
ability to use new metabolites.

23.  The application of conjugate plasmid recombination technology
is employed for strain improvement of Lactococcus bacteria
starter cultures in the dairy industry.
 The improved Lactococcus strain resistant to Bacteriophage
infection as well as antibiotic resistant.

24. Transduction:

25. Transduction:
Two types:
General transduction:
Specialized transduction:

26. Transduction:
 Transduction is the transfer of bacterial DNA from one bacterial
cell to another by means of a bacteriophage.
 Two types:
General transduction:
Specialized transduction:
 In general transduction, any part of the host DNA is transferred by
Bacteriophage.
 In specialized transduction, a specific gene is transferred by
Bacteriophage.
 The method is a well-established research tool in genetic
recombination.

27. Genetic Engineering:

28. Genetic engineering, also known as recombinant DNA
technology, molecular cloning or gene cloning.
 Recombinant DNA Technology involves isolation of genes
from an organism, amplified of gene and insertion of gene
into another organism.

30.  By using Restriction endonucleases and ligases, investigators can
cut and insert DNA in to suitable vector (plasmid or phase).

31.  Genetic engineering involves the following steps:
1. Isolate the desired gene (DNA fragment) from the donor
cells.
2. Isolate the vector (a plasmid or a phage).
3. Cleave the vector, align the donor DNA with the vector, and
insert the gene into the vector.
4. Introduce the new plasmid into the host cell by
transformation or, if a viral vector is used, by infection.
5. Select the new recombinant strains that express the desired
characteristics.

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