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

2. Some applications of recombiant DNA technology in Biotechnology
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 Steps (not in order)
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

11. Pancreas Vector Plasmid Gene Host Sticky ends
Hormone
Pancreas
Vector
Plasmid
Transformation
Gene
Insulin
Host
Recombinant DNA
Restriction
enzyme
DNA ligase
Sticky ends

12. What else?

13. Recombinant enzymes for food processing

14. recombinant DNA technology steps in
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. single polypeptide chain of 323 amino acids
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
the chymosin content of the rennin causes a specific and rapid cleaveage of the kappa-casein component of the casein micelles. This protein stabilises the micelles and after cleavage, the casein proteins precipitate under the influence of the calcium ions. Chymosin specifically recognises the sequence from His 98 to Lys 111 and cleaves the peptide bond between Phe 105 and Met 106 in the kappa-casein chain:

19. 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

21. 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
Current strain improvement strategies have already contributed to creating more efficient and safer enzyme production strains. This trend will undoubtedly continue as the knowledge about the genetic make-up of microorganisms used for enzyme production expands and new genetic techniques emerge.

22. 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

23. 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)

24. 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)

25. 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.

26. Four steps in Golden Rice Technology

27. How gene is introduced into rice:

28. 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. 

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

30. 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.

31. 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.

32. 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