Biotechnology- Principles and Processes

Author: lovelybotany  //  Category: BITECHNOLOGY : PRINCIPLES AND PROCESSES

Biotechnology deals with technique of using live organisms or enzymes from organisms to produce products and processes useful to humans. It includes the use of genetically modified organisms to achieve the same on a large scale, invitro fertilisation leading to a test tube baby, synthesis and use of a gene , developing a DNA vaccine or correcting a defective gene etc.

European Federation of Biotechnology ( EFB ) gives a defenition to Biotechnology as ” The integration of natural science and organisms , cells, parts thereof , and molecular analogues for products and services ”

Principles of Biotechnology

  • Genetic engineering: Techniques to alter the chemistry of genetic material ( DNA and RNA ) , to introduce these into host organisms and thus change the phenotype of host organisms.
  • Maintenance of sterile conditions in chemical engineering processes for growing single types of microbes for the manufacture of products like antibiotics , vaccines, enzymes etc.
What are the advantages of Biotechnology over Traditional Hybridization?
It often lead to the inclusion and multiplication of undesirable genes along with desired genes.
Biotechnology overcomes this limitation by isolating and introducing one or a set of desirable genes.
What will be the fate of DNA transferred to an alien organism with out ‘Origin of replication’ ?
Multiplication of alien piece of DNA is possible as it integrates with a specific sequence called  ’Origin of replication’ which is responsible for the replication and multiplication with in the host organism.

 

>Figure 1. The process of making recombinant DNA, as pioneered by Paul Berg.Figure 1. The process of making recombinant DNA, as pioneered by Paul Berg.
The Construction of  first Recombinant DNA by Stanley Cohen and Herbert Boyer in 1972. The steps are given below :-

 

Figure 2. The insertion of recombinant DNA so that the foreign DNA will replicate naturally, as pioneered by Herbert Boyer and Stanley Cohen.Figure 2. The insertion of recombinant DNA so that the foreign DNA will replicate naturally, as pioneered by Herbert Boyer and Stanley Cohen.
1. Cutting the gene encoding antibiotic resistance
2. Linking with the native plasmid of Salmonella typhimurium.
3. Cutting is possible by restriction enzymes
4. Linking by DNA ligase.
5. Recombinant DNA  can be transferred to another bacterium E. coli, for replication and production of multiple copies, called Gene cloning.
Tools of Recombinant DNA Technology
A. Restriction enzymes.
B. Polymerase enzymes.
C. Ligases
D. Vectors.
E. Host organism.
A. Restriction enzymes.
  • Restriction enzymes were isolated from E.coli  in 1963, one restricting the growth by adding methyl groups and other cutting DNA.the later was termed as Restriction Endonuclease.
  • Hind II is the first restriction enzyme isolated , characterised five years later.
  • Hind II cuts by identifying particular six pairs of bases known as Recognition Sequence
  • 900 restriction enzymes were identified from 230 strains of bacteria.
  • The convention for naming the enzyme is that the first letter comes from the genus and second letter from the species from which it isolated. EcoR1 comes from Escherichia coli RY13. In EcoR1 ,  the letter R is derived from the name of strain.
  • Restricion enzymes belong to a large class of enzymes called nucleases that is exonucleases and endonucleases.
  • Exonucleases make cuts at the ends of DNA sequences.
  • Endonucleases make cuts at specific positions with in the DNA.
  • Each restriction enzymes acts on specific recognition sequence called Palindromic nucleotide sequences in the DNA.
  • Palindromic nucleotide sequences are sequence of base pairs that reads same on two strands when orientation of reading   is kept same.
  • Cut occurs a little away from the centre between the same base pairs and that leaves single stranded portions called Sticky ends on each strand . They can form hydrogen bonds with their counter parts, and DNA ligase can easily act on it.
  • When cut by similar restriction enzymes leaves similar sticky ends.

 

NCERT Solutions

Question 1:

Can you list 10 recombinant proteins which are used in medical practice? Find out where they are used as therapeutics (use the internet).

Recombinant proteins are obtained from the recombinant DNA technology. This technology involves the transfer of specific genes from an organism into another organism using vectors and restriction enzymes as molecular tools.

Ten recombinant proteins used in medical practice are −

Recombinant protein

Therapeutic use

1. Insulin Treatment for type I diabetes mellitus
2. Interferon-α Used for chronic hepatitis C
3. Interferon -β Used for herpes and viral enteritis
4. Coagulation factor VII Treatment of haemophilia A
5. Coagulation factor IX Treatment of haemophilia B
6. DNAase I Treatment of cystic fibrosis
7. Anti-thrombin III Prevention of blood clot
8. Interferon B. For treatment of multiple sclerosis
9. Human recombinant growth hormone For promoting growth in an individual
10. Tissue plasminogen activator  Treatment of acute myocardial infection

 

Question 2:

Make a chart (with diagrammatic representation) showing a restriction enzyme, the substrate DNA on which it acts, the site at which it cuts DNA and the product it produces.

  • Answer
  • The name of the restriction enzyme is Bam H 1.

Question 3:

From what you have learnt, can you tell whether enzymes are bigger or DNA is bigger in molecular size? How did you know?

Enzymes are smaller in size than DNA molecules. This is because DNA contains genetic information for the development and functioning of all living organisms. It contains instructions for the synthesis of proteins and DNA molecules. On the other hand, enzymes are proteins which are synthesised from a small stretch of DNA known as ‘genes’, which are involved in the production of the polypeptide chain.

What would be the molar concentration of human DNA in a human cell? Consult your teacher.

The molar concentration of human DNA in a human diploid cell is as follows:

⇒ Total number of chromosomes × 6.023 × 1023

⇒ 46 × 6.023 × 10­­­23

⇒ 2.77 ×1018 Moles

Hence, the molar concentration of DNA in each diploid cell in humans is 2.77 × 10 23 moles.

Question 5:

Do eukaryotic cells have restriction endonucleases? Justify your answer.

No, eukaryotic cells do not have restriction endonucleases. This is because the DNA of eukaryotes is highly methylated by a modification enzyme, called methylase. Methylation protects the DNA from the activity of restriction enzymes .These enzymes are present in prokaryotic cells where they help prevent the invasion of DNA by virus.

Question 6:

Besides better aeration and mixing properties, what other advantages do stirred tank bioreactors have over shake flasks?

The shake flask method is used for a small-scale production of biotechnological products in a laboratory. On the other hand, stirred tank bioreactors are used for a large-scale production of biotechnology products.

Stirred tank bioreactors have several advantages over shake flasks:

(1) Small volumes of culture can be taken out from the reactor for sampling or testing.

(2) It has a foam breaker for regulating the foam.

(3) It has a control system that regulates the temperature and pH

Question 7:

Collect 5 examples of palindromic DNA sequences by consulting your teacher. Better try to create a palindromic sequence by following base-pair rules.

The palindromic sequence is a certain sequence of the DNA that reads the same whether read from 5 → 3’ direction or from 3→ 5direction. They are the site for the action of restriction enzymes. Most restriction enzymes are palindromic sequences.

Five examples of palindromic sequences are:

Question 8:

Can you recall meiosis and indicate at what stage a recombinant DNA is made?

Meiosis is a process that involves the reduction in the amount of genetic material. It is two types, namely meiosis I and meiosis II. During the pachytene stage of prophase I, crossing over of chromosomes takes place where the exchange of segments between non-sister chromatids of homlogous chromosomes takes place. This results in the formation of recombinant DNA.

 

Question 9:

Can you think and answer how a reporter enzyme can be used to monitor transformation of host cells by foreign DNA in addition to a selectable marker?

A reporter gene can be used to monitor the transformation of host cells by foreign DNA. They act as a selectable marker to determine whether the host cell has taken up the foreign DNA or the foreign gene gets expressed in the cell. The researchers place the reporter gene and the foreign gene in the same DNA construct. Then, this combined DNA construct is inserted in the cell. Here, the reporter gene is used as a selectable marker to find out the successful uptake of genes of interest (foreign genes). An example of reporter genes includes lac Z gene, which encodes a green fluorescent protein in a jelly fish.

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