Applications of Recombinant DNA Technology in Biotechnology

 
 
Lecture 3
Recombinant DNA Technology and Biotechnology II
 
FE314
-
 Biotechnology
Spring 
201
6
 
 
Some  applications of recombiant DNA
technology in Biotechnology
*Diabates-Insulin production(medical
biotechnology)
*Enzyme production (Food Biotechnology)
*Golden rice(Agricultural and Food
Biotechnology)
 
Example: Production of Insulin
(Medical Biotechnology)
 
Diatebes and insulin requirement
 
Understand what Diabetes is and how it
affects the person
Understand how insulin can be produced
by recombinant DNA technology
 
Diabetes mellitus
 (
DM
),
 
Diabetes mellitus
 (
DM
), commonly referred to
as 
diabetes
, is a group of metabolic diseases
 
in
which there are high blood sugar
 
levels over a
prolonged period.
Diabetes is due to either the pancreas not
producing enough insulin
 
or the cells of the
body not responding properly to the insulin
produced
Insulin
– what is it exactly?
 
A 
hormone
(chemical
messenger) that
allows glucose to
pass from the
blood into your
cells.
 
How it works
Recombinant DNA technology
 
Joining together DNA molecules from two
different species.  This is then inserted into
a host organism which can produce new
genetic combinations that are useful to us
Bacteria – 
E. coli
 
Transformation
 
Steps (not in order)
 
2.
The gene for insulin is isolated and
removed from human DNA leaving it with sticky ends
 
4.
A plasmid from the bacteria E coli is removed
and cut open using restriction enzymes
 
1.
The insulin gene is inserted into the bacterial plasmid using DNA ligase
 
3.
The recombinant plasmid is
taken up by the bacteria by transformation
 
5.
The bacteria reproduces, making copies of the gene
each time, allowing lots of insulin to be produced
Key words
Isolated gene
 
Sticky ends
  
Vector
 
Plasmid
 
Restriction enzyme
 
 
DNA ligase
 
Recombinant plasmid
  
Transformation
What else?
 
 
Recombinant enzymes for food
processing
 
 
Food-processing enzymes from recombinant microorganisms
 
 
food processing and in the production of food ingredients
Enzymes traditionally isolated from culturable microorganisms, plants, and mammalian
tissues are often not well-adapted to the conditions used in modern food production
methods
    recombinant DNA technology steps in
 
 manufacture novel enzymes suitable for specific food-processing conditions.
 
                                                  How ?
 by screening microorganisms sampled from diverse environments
by modification of known enzymes using modern methods of
protein engineering
 
                                                 Advantages
Improvement of microbial production strains
 increase enzyme yield by deleting native genes encoding extracellular proteases
fungal production strains have been modified to reduce or eliminate their potential for
production of toxic secondary metabolites
 
 
 
 
Improved pectinase production in 
Penicillium griseoroseum 
recombinant strains.
 
Experiments have done to obtain a recombinant organism that will be having the ability to
obtain pectin lyase (PL) and polygalacturonase (PG) and for that 
P
enicillium griseoroseum 
that
produced both PL &PG simultaneously.
 
Firstly a strain that was reported to produce high concentration of PL was taken.
It was then transformed using pAN52pgg2 plasmid which was having a foreign gene of PG of P.
grieoroseum and it was having a promoter from 
Aspergillus nidulans
The newly transformed P. grieoroseum T20 when checked was producing higher concentrations
of both PG and PL, around 143 folds higher PL, and 15 folds greater PG.
This recombinant strain uses carbon sources of low costs that is very economical
The enzyme preparation commercially available is free of cellulolytic and proteolytic activities.
This is an efficient system that uses 
P. griseoroseum 
to express and secrete proteins.
 
 
 
 
Chymosin
 
Also known as rennin
single polypeptide chain of 323 amino acids
2 types  differ only by one amino acid
Chymosin A (aspartic acid residue at position 286)
Chymosin B (glycine residue at position 286 )
‡
 
Main coagulating enzyme
 found in rennet which is used
 extensively in cheese
 production.
hydrolyses a specific site in
 kappa-casein of milk
 
kappa-casein acts as micelle stabilizer
‡Precipitation of insoluble constituents
 
 
 
 
 
 
 
                            Search for an alternative rennet
increasing demand
 
shortage of calf stomachs
 
ethical issues with animal slaughtering
 
Kluyveromyces lactis
 
increase Chymosin production has been
 made through expression of calf Chymosin
gene in recombinant K. lactis
                                  Advantages of K. Lactis
Non toxicogenic GRAS microorganism approved by US FDA
‡Unlike p. Pastoris it does not require methanol to induce protein
secretion
‡Unlike E. coli it doesn't secrete the expressed protein enclosed in
inclusion bodies
 
 
 
 
 
 
Recombinant bovine Chymosin is the most frequently used
Chymosin in the industry
new sources of recombinant Chymosin, such as goat, camel, or
buffalo, are now available.
 
When 
compared
 with other 3 enzymes
 
Recombinant goat Chymosin exhibited the 
best catalytic efficiency
recombinant goat Chymosin exhibited the 
best specific proteolytic
activity,
recombinant goat Chymosin exhibited a 
wider pH range of action,
recombinant goat Chymosin exhibited a 
lower glycosylation degree
 
 
 
comparative study of 4 different recombinant chymosins
 
 
Golden rice-Agricultural and food
biotechnology
Vitamin A deficiency:  The Problem
 
Weakens the immune system
 
Can lead to blindness which
increases the risk of death
 
400 million poor in rice-based societies are
Vitamin A deficient.
500,000 children go blind per year.
(UNICEF)
1.15 million VAD-precipitated deaths among
children world wide.
(UNICEF)
Rice is the main staple crop for most of these
children, but rice lack pro-vitamin A and other
micronutrients.
(UNICEF)
 
Vitamin A deficiency: The Solution
 
Golden Rice
Development by 
Ingo 
Potrykus and 
Peter 
Beyer
 Contains a gene from maize or daffodil plants and
common soil bacterium (
Erwinia
)
 
 
Who Began the Golden
Rice Project?
 
Started in 1982 by Ingo Potrykus-Professor
emeritus of the Institute for Plant
Sciences
,Switerland
Peter Beyer-Professor of Centre for Applied
Biosciences, Uni. Of Freiburg, Germany
Funded by the Rockefeller Foundation, the
Swiss Federal Institute of Technology, and
Syngenta, a crop protection company.
 
Four steps in Golden Rice Technology
How gene is introduced into rice:
 
MECHANISM:
 
 
 
 
Golden rice was created by transforming rice with only two beta-carotene biosynthesis genes:
 
psy
 (phytoene synthase) from daffodil (
Narcissus pseudonarcissus
)
 
crtI
 (carotene desaturase) from the soil bacterium 
Erwinia uredovora
 
(The insertion of a 
lyc
 (lycopene cyclase) gene was thought to be needed, but further research
showed it is already being produced in wild-type rice endosperm.)
 
The 
psy
  and 
crtI
  genes were transformed into the rice nuclear genome and placed under the
control of an endosperm-specific promoter, so they are only expressed in the endosperm. The
bacterial 
crtI
  gene was an important inclusion to complete the pathway, since it can catalyze
multiple steps in the synthesis of carotenoids up to lycopene, while these steps require more than
one enzyme in plants.
 
 
The Golden Rice Solution
 
Daffodil gene
 
Bacterial gene
 
-Carotene Pathway Genes Added
 
Why rice?
 
Other plants, such as sweet potatoes have varieties that are either
Other plants, such as sweet potatoes have varieties that are either
rich (orange-fleshed) or poor (white fleshed) in pro-vitamin A
rich (orange-fleshed) or poor (white fleshed) in pro-vitamin A
It is difficult to manage even two times meal for the poor people.
It is difficult to manage even two times meal for the poor people.
 
Global staple food. Cultivated
Global staple food. Cultivated
for over 10,000 years
for over 10,000 years
Rice provides as much as 80
Rice provides as much as 80
percent or more of the daily
percent or more of the daily
caloric intake of 3 billion
caloric intake of 3 billion
people, which is half the world’s
people, which is half the world’s
population
population
 
 
 
 
Interesting fact about golden rice:
 
The yellow colour of golden rice is due to the
presence of 
β
- carotenoid.
 
 In one transgenic line, the 
β
-carotene content
was as high as 85% of the total carotenoid
present in the grain.
 
Negative impact:
 
Health
May cause allergies or fail to perform desired effect
Supply does not provide a substantial quantity as the
recommended daily intake
Environment
Loss of Biodiversity. May become a gregarious weed and
endanger the existence of natural rice plants
Genetic contamination of natural, global staple foods
Culture
Some people prefer to cultivate and eat only white rice
based on traditional values and spiritual beliefs
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"Explore the various applications of recombinant DNA technology in biotechnology, including insulin production for diabetes, enzyme production in food biotechnology, and the development of Golden Rice. Understand the process of insulin production using recombinant DNA technology and the significance of insulin in managing diabetes mellitus."


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  1. FE314- Biotechnology Spring 2016 Lecture 3 Recombinant DNA Technology and Biotechnology II

  2. Some applications of recombiant DNA technology in Biotechnology *Diabates-Insulin production(medical biotechnology) *Enzyme production (Food Biotechnology) *Golden rice(Agricultural and Food Biotechnology)

  3. Example: Production of Insulin (Medical Biotechnology)

  4. Diatebes and insulin requirement Understand what Diabetes is and how it affects the person Understand how insulin can be produced by recombinant DNA technology

  5. Diabetes mellitus (DM), Diabetes mellitus (DM), commonly referred to as diabetes, is a group of metabolic diseases in which there are high blood sugar levels over a prolonged period. Diabetes is due to either the pancreas not producing enough insulin or the cells of the body not responding properly to the insulin produced

  6. Insulin what is it exactly? A hormone (chemical messenger) that allows glucose to pass from the blood into your cells.

  7. How it works

  8. Recombinant DNA technology Joining together DNA molecules from two different species. This is then inserted into a host organism which can produce new genetic combinations that are useful to us

  9. Bacteria E. coli Transformation

  10. Key words Sticky ends Restriction enzyme Isolated gene Plasmid Recombinant plasmid Vector DNA ligase Transformation Steps (not in order) 1.The insulin gene is inserted into the bacterial plasmid using DNA ligase 2.The gene for insulin is isolated and removed from human DNA leaving it with sticky ends 3.The recombinant plasmid is taken up by the bacteria by transformation 4.A plasmid from the bacteria E coli is removed and cut open using restriction enzymes 5.The bacteria reproduces, making copies of the gene each time, allowing lots of insulin to be produced

  11. Pancreas PLASMID Gene Recombinant DNA DNA ligase Restriction enzyme Sticky ends

  12. What else?

  13. Recombinant enzymes for food processing

  14. Food-processing enzymes from recombinant microorganisms food processing and in the production of food ingredients Enzymes traditionally isolated from culturable microorganisms, plants, and mammalian tissues are often not well-adapted to the conditions used in modern food production methods recombinant DNA technology steps in manufacture novel enzymes suitable for specific food-processing conditions. How ? by screening microorganisms sampled from diverse environments by modification of known enzymes using modern methods of protein engineering Advantages Improvement of microbial production strains increase enzyme yield by deleting native genes encoding extracellular proteases fungal production strains have been modified to reduce or eliminate their potential for production of toxic secondary metabolites

  15. Improved pectinase production in Penicillium griseoroseum recombinant strains. Experiments have done to obtain a recombinant organism that will be having the ability to obtain pectin lyase (PL) and polygalacturonase (PG) and for that Penicillium griseoroseum that produced both PL &PG simultaneously. Firstly a strain that was reported to produce high concentration of PL was taken. It was then transformed using pAN52pgg2 plasmid which was having a foreign gene of PG of P. grieoroseum and it was having a promoter from Aspergillus nidulans The newly transformed P. grieoroseum T20 when checked was producing higher concentrations of both PG and PL, around 143 folds higher PL, and 15 folds greater PG. This recombinant strain uses carbon sources of low costs that is very economical The enzyme preparation commercially available is free of cellulolytic and proteolytic activities. This is an efficient system that uses P. griseoroseum to express and secrete proteins.

  16. Chymosin

  17. Also known as rennin single polypeptide chain of 323 amino acids 2 types differ only by one amino acid Chymosin A (aspartic acid residue at position 286) Chymosin B (glycine residue at position 286 ) Main coagulating enzyme found in rennet which is used extensively in cheese production. hydrolyses a specific site in kappa-casein of milk kappa-casein acts as micelle stabilizer Precipitation of insoluble constituents

  18. Search for an alternative rennet increasing demand shortage of calf stomachs ethical issues with animal slaughtering Kluyveromyces lactis increase Chymosin production has been made through expression of calf Chymosin gene in recombinant K. lactis Advantages of K. Lactis Non toxicogenic GRAS microorganism approved by US FDA Unlike p. Pastoris it does not require methanol to induce protein secretion Unlike E. coli it doesn't secrete the expressed protein enclosed in inclusion bodies

  19. comparative study of 4 different recombinant chymosins Recombinant bovine Chymosin is the most frequently used Chymosin in the industry new sources of recombinant Chymosin, such as goat, camel, or buffalo, are now available. When compared with other 3 enzymes Recombinant goat Chymosin exhibited the best catalytic efficiency recombinant goat Chymosin exhibited the best specific proteolytic activity, recombinant goat Chymosin exhibited a wider pH range of action, recombinant goat Chymosin exhibited a lower glycosylation degree

  20. Golden rice-Agricultural and food biotechnology Vitamin A deficiency: The Problem Weakens the immune system Can lead to blindness which increases the risk of death

  21. 400 million poor in rice-based societies are Vitamin A deficient. 500,000 children go blind per year.(UNICEF) 1.15 million VAD-precipitated deaths among children world wide.(UNICEF) Rice is the main staple crop for most of these children, but rice lack pro-vitamin A and other micronutrients.(UNICEF)

  22. Vitamin A deficiency: The Solution Golden Rice Development by Ingo Potrykus and Peter Beyer Contains a gene from maize or daffodil plants and common soil bacterium (Erwinia)

  23. Who Began the Golden Rice Project? Started in 1982 by Ingo Potrykus-Professor emeritus of the Institute for Plant Sciences,Switerland Peter Beyer-Professor of Centre for Applied Biosciences, Uni. Of Freiburg, Germany Funded by the Rockefeller Foundation, the Swiss Federal Institute of Technology, and Syngenta, a crop protection company.

  24. Four steps in Golden Rice Technology

  25. How gene is introduced into rice:

  26. MECHANISM: Golden rice was created by transforming rice with only two beta-carotene biosynthesis genes: psy (phytoene synthase) from daffodil (Narcissus pseudonarcissus) crtI (carotene desaturase) from the soil bacterium Erwinia uredovora (The insertion of a lyc (lycopene cyclase) gene was thought to be needed, but further research showed it is already being produced in wild-type rice endosperm.) The psy and crtI genes were transformed into the rice nuclear genome and placed under the control of an endosperm-specific promoter, so they are only expressed in the endosperm. The bacterial crtI gene was an important inclusion to complete the pathway, since it can catalyze multiple steps in the synthesis of carotenoids up to lycopene, while these steps require more than one enzyme in plants.

  27. The Golden Rice Solution -Carotene Pathway Genes Added Geranylgeranyl diphosphate Phytoene synthase Daffodil gene Phytoene Vitamin A Pathway is complete and functional Bacterial gene carotene desaturase Lycopene Lycopene cyclase -carotene (vitamin A precursor) Golden Rice

  28. Why rice? Global staple food. Cultivated for over 10,000 years Rice provides as much as 80 percent or more of the daily caloric intake of 3 billion people, which is half the world s population View Image Other plants, such as sweet potatoes have varieties that are either rich (orange-fleshed) or poor (white fleshed) in pro-vitamin A It is difficult to manage even two times meal for the poor people.

  29. Interesting fact about golden rice: The yellow colour of golden rice is due to the presence of - carotenoid. In one transgenic line, the -carotene content was as high as 85% of the total carotenoid present in the grain.

  30. Negative impact: Health May cause allergies or fail to perform desired effect Supply does not provide a substantial quantity as the recommended daily intake Environment Loss of Biodiversity. May become a gregarious weed and endanger the existence of natural rice plants Genetic contamination of natural, global staple foods Culture Some people prefer to cultivate and eat only white rice based on traditional values and spiritual beliefs

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