Biotechnology Principles and Process Question Answers: NCERT Class 12 Biology

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 −

(a) Origin of replication −Origin of replication is defined as the DNA sequence in a genome from where replication initiates. The initiation of replication can be either uni-directional or bi-directional. A protein complex recognizes the ‘on’ site, unwinds the two strands, and initiates the copying of the DNA.

(b) Bioreactors − Bioreactors are large vessels used for the large-scale production of biotechnology products from raw materials. They provide optimal conditions to obtain the desired product by providing the optimum temperature, pH, vitamin, oxygen, etc. Bioreactors have an oxygen delivery system, a foam control system, a PH, a temperature control system, and a sampling port to obtain a small volume of culture for sampling.

(c) Downstream processing − Downstream processing is a method of separation and purification of foreign gene products after the completion of the biosynthetic stage. The product is subjected to various processes in order to separate and purify the product. After downstream processing, the product is formulated and is passed through various clinical trials for quality control and other tests.

 (a) PCR: - Polymerase chain reaction (PCR) is a technique in molecular biology to amplify a gene or a piece of DNA to obtain its several copies. It is extensively used in the process of gene manipulation. The process involves in-vitro synthesis of sequences using a primer, a template strand, and a thermostable DNA polymerase enzyme obtained from a bacterium, called Thermus aquaticus. The enzyme utilizes building blocks dNTPs (deoxynucleotides) to extend the primer. In the first step, the double stranded DNA molecules are heated to a high temperature so that the two strands separate into a single stranded DNA molecule. This process is called denaturation. Then, this ssDNA molecule is used as a template strand for the synthesis of a new strand by the DNA polymerase enzyme and this process is called annealing, which results in the duplication of the original DNA molecule. This process is repeated over several cycles to obtain multiple copies of the rDNA fragment.

(b) Restriction enzymes are molecular scissors used in molecular biology for cutting DNA sequences from a specific site. It plays an important role in gene manipulation. The enzymes recognize a specific six-box pair sequence known as the recognition sequence and cut the sequence at a specific site. For example, the recognition site for enzyme ECORI is as follows:

Restriction enzyme are categorized into two types −

(i) Exonuclease − It is a type of restriction enzyme that removes the nucleotide from either 5^’ or 3^’ ends of the DNA molecule.

(ii) Endonuclease − It is a type of restriction enzyme that makes a cut within the DNA at a specific site. This enzyme acts as an important tool in genetic engineering. It is commonly used to make a cut in the sequence to obtain DNA fragments with sticky ends, which are later joined by enzyme DNA ligase.

(c) Chitinase − Chitinase is a class of enzymes used for the degradation of chitin, which forms a major component of the fungal cell wall. Therefore, to isolate the DNA enclosed within the cell membrane of the fungus, enzyme chitinase is used to break the cell for releasing its genetic material.

(a) Plasmid DNA and chromosomal DNA

(b) RNA and DNA

(c) Exonuclease and Endonuclease

The name of the restriction enzyme is Bam H 1.

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.

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

⇒ Total number of chromosomes × 6.023 × 10²³

⇒ 46 × 6.023 × 10_­­­²³

⇒ 2.77 ×10¹⁸ Moles

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

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.

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.

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

Five examples of palindromic sequences are:

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.

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.

In the selection of recombinant plasmids, antibiotic resistant genes are used as selectable markers which is very difficult. Insertion inactivation requires the two alternative plating of culture media and possess alternative selective markers that have been developed to facilitate the methodology; these alternative methods are used to differentiate between recombinants and non- recombinants. In the presence of chromogenic substrate non- recombinants produces colour. And, recombinant- DNA is integrated into the coding sequence of β- galactosidase enzyme, through which the enzyme get inactivated, is known as insertional inactivation. Non- recombinant plasmid of bacteria gives blue colour in the presence of chromogenic substrate and the recombinant plasmid leads into insertional inactivation of β- galactosidase enzyme so that colour will not develop and hence, the recombinant colonies are identified.

Bioreactors are the large vessels in which raw materials are biologically converted into the specific products. Recombinant DNA leads to the synthesis of desired proteins which is also known as recombinant proteins under the controlled conditions and large scale production of these products are carried out by bioreactors. Antibiotics presence makes sure that bacteria retains the plasmid which contains antibiotic resistant genes.

In the absence of antibiotics, there will be no pressure to continue the plasmid on the recombination processes and as a result it will not maintain the high copy number of plasmids which made due to the metabolic pressure. And hence lose the continuity of making the high copy number of plasmids.

A. Denaturation

B. Annealing

C. DNA synthesis or Extension of primers

PCR is a chain reaction because of newly synthesized DNA acts as templates for further DNA formation in the form of cycles. In each cycle, the duplex DNA strands are separated by the heat denaturation at 93- 95◦C and then it is cooled so that synthetic DNA primers can anneal to the complementary segments on each strand. The annealing temperature usually occurs at about 50- 70◦C; this annealing temperature should be low that enables the hybridization between primer and the template strand and should be high as to prevent the mismatched hybridization. Then DNA synthesis occurs at about 70-75◦C temperature and then this extension is obtained by DNA polymerase enzyme. The process is then repeated several times and the unit- length of the cycle doubles with every cycle.

1. Bam HI
2. Pst I
3. amp^R (ampicillin resistance gene)

This diagram is the representation of pBR 322 vector, in which p stands for plasmid and BR stands for their developers Bolivar and Rodriguez; and ‘322’ defines the plasmid identification number developed by other scientists in the same laboratory. This has tetracycline and ampicillin resistance gene on its host and have cleavage sites for Pst I, EcoRI, HindIII, Bam HI, SalI and ClaI, Pvu II. Ampicillin resistance gene inhibits bacterial cell wall synthesis by disrupting cross linkage of peptidoglycans.

Ti (tumour- inducing) plasmid which is present in Agrobacterium tumefaciens is used as vector for plants that has been considered as natural genetic engineers for transforming plants. It causes a crown gall disease in dicot plants by transferring a segment of Transfer- DNA from Ti- plasmid into nuclear genome of plant cells. When the plant molecular biologists have started using Ti plasmid as vectors when it is disarmed, then it is no more harmful for the plants and they don’t cause any disease.

Structure of Ti Plasmid:-

This Ti plasmid of Agrobacterium tumefaciens contains ori (origin of replication), bundle or cluster of virulence genes (helps to encode proteins that are required for T- DNA processing) and the T- DNA. And, T- DNA is flanked by improper direct repeats which are known as left and right border (for transfer and integration to the plant genome) sequences. T- DNA carries genes for the production of phytohormones like auxin, cytokinin and opine; and these genes are transcribed and translated in the plant cells. Their overproduction at the site of infection leads to multiplication of wound cells into the gall tumour and the opines are used as the carbon and nitrogen source which are being used by the Agrobacterium.

Diagram:-

Agrobacterium tumefaciens genetic transformation is a complex process which involves the Agrobacterium and the host. It involves T- DNA, virulence genes and T4SS (for macromolecules exportation). This process occurs by various virulence genes and every gene has their specific role in this process. In short, Firstly bacteria will attach to the plant cell which facilitates the interaction between the plant inducers. And this activates the transcription activator virulence then induce the rest of the virulence genes that are present on Ti- plasmid. T- DNA is transferred into the nucleus and integrates into the plant cell chromosome through the T4SS (type IV secretion system) which is also known as Mpf (Mating pair formation) apparatus that forms a pore or channel through which the DNA or protein get transferred from donor to the recipient cell.

Diagram:-

Diagram of bioreactor:-

The restriction enzyme cuts double helical segments which contains particular nucleotide sequence which is cut down by the restriction endonuclease enzyme at specific recognition sequence. If the restriction enzyme doesn’t cut the DNA at specific position, it will not produce any sticky ends (cleavage does not falls on symmetrical axis, as in results restriction fragments will give sticky ends) and recombination will also not occur. Instead of giving sticky ends it will produce blunt ends when it cut down from the random position.

Plasmid DNA shows one band after cutting by restriction endonuclease because it is a circular shaped DNA. When it is cut down then it will open up and show linear DNA fragment. And when linear DNA is cut down with specific restriction endonuclease enzyme it will divide into two fragments after cleaving and hence show two bands when we put this on the agarose gel electrophoresis. 

Size of the DNA fragments depends on cleavage site or its position that is from when it cuts the DNA molecule.

Gel electrophoresis technique is used to separate the DNA fragments, in which matrix, agarose gel is used. DNA fragments moves towards the anode according to their molecular size through this gel, and then the smaller fragments get separated from the larger fragments. And, these separated DNA fragments can be only observed after staining with the solution of ethidium bromide. Bright orange colour bands will appear only under the UV- light. Ethidium bromide is the most commonly used fluorescent dyes which is used to intercalate between bases of nucleic acids and their fragments. It is used because of its short staining time.

Selectable marker encodes a product which allows the artificial selection and growth of transformed or transfected cells. The cloning vector needs the presence of selectable marker used to identify and eliminate the non- transformant cell. Antibiotic resistance gene are mostly used as selectable markers and as well as antibiotics. A host cell strain is chosen which will react to the particular antibiotics like ampicillin, tetracycline, kanamycin etc. which are been used in transformation process.

So, if there will be no selectable marker, no identification of transformed cell that contains DNA molecule will occur.  

Note*:- Ampicillin:- it inhibits the bacterial cell wall synthesis.

              Tetracycline:- It inhibits the binding of aminoacyl tRNA to the 30S ribosomal subunit.

              Kanamycin:- it inactivates the translation process.

A mixture of DNA fragments was electrophoresed in agarose gel and after staining with the ethidium bromide, no bands were appeared. There could be many reasons for that and these are following:-

1. May be the staining concentration is not enough to stain the DNA fragments so that the bands appears.
2. Isolation process can be degraded and no enzymes worked on break down of cell envelope.
3. By improper cutting or it could be occur because of endonuclease when cuts on random position or when exonuclease and endonuclease cuts at the same time; or can be loaded in the DNA sample on the agarose gel. 

CaCl₂ is used to increase the efficiency of DNA uptake for the bacterial transformation. It leads to the DNA precipitation onto the surface of the cells and the ice cold salt contains CaCl₂ follows heat shock at 42◦C, is responsible for the changes in the cell wall which improves DNA binding. The divalent Ca²⁺ ions creates transient pores on the bacterial cell wall through which the entry of foreign DNA is facilitated into the cells of bacteria.