Download Diabetes mellitus

FE314- Biotechnology
Spring 2016
Lecture 3
Recombinant DNA Technology and Biotechnology II
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
Key words
Isolated gene
Sticky ends
Vector
Plasmid
Restriction enzyme
DNA ligase
Recombinant plasmid
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
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 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.
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
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
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
-Carotene Pathway Genes Added
Geranylgeranyl diphosphate
Phytoene
synthase
Daffodil gene
Vitamin A
Pathway
is complete
and functional
Phytoene
Bacterial gene
carotene desaturase
Lycopene
Lycopene cyclase
Golden
Rice
 -carotene
(vitamin A precursor)
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
• 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.
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