Thinking Process

Dough is formed by fermentation by bacteria.

Answer

(b) Inoculation of kneaded flour with baker yeast; Saccharomyces cerevisiae produce $\mathrm{CO} _{2}$ during the process of fermentation, causes puffing up of the dough and make it soft and spongy. It is used to make foods like idli, dosa, bread, etc.

Answer

(b) Restriction enzymes belongs to a class of enzymes called nucleases and are of two types

(i) Exonucleases remove nucleotides from the ends of the DNA.

(ii) Endonucleases make cuts at specific positions within the DNA.

DNA ligase is a sealing enzymes (also called as genetic gum), which is responsible for joining of two individual fragments of DNA, whereas Hind II is first discovered restriction endonuclease enzyme.

Answer

(a) Transduction is the process by which genetic material (DNA) is transferred from one bacterium to another through the mediation of a vector, like virus

Other option are incorrect because bacterial conjugation is the transfer of genetic material (plasmid) between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells.

Transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membranes.

Translation is the process in which cellular ribosomes create proteins. It is a part of the process of gene expression.

Thinking Process

Gel electrophoresis is a technique for separating DNA fragments based on their size.

Answer

(d) The separated DNA fragments (by the process of gel electrophoresis) are visualised after staining the DNA with ethidium bromide followed by exposure to UV-radiation. These fragments are seen as orange coloured bands.

Answer

(b) The restriction enzymes are called ‘molecular scissors’ and are responsible for cutting DNA. Restriction enzymes belong to a class of enzymes called nucleases.

They are of two types

(i) Exonucleases Cut DNA at the ends

(ii) Endonucleases Make cuts at specific positions within the DNA.

These enzymes are present in bacteria to provide a type of defense mechanism called the ‘restriction modification system’.

This system consist of two components, restriction enzyme and modification enzyme. The term ‘restriction’ refer to the function of these enzyme in restricting the propagation of foreign DNA of bacteriophage in host bacterium, i.e., cutting of DNA, at specific position only.

Answer

(d) Restriction enzymes and DNA ligases can be used to make a stable recombinant DNA molecule, with DNA fragments that has been spliced together from two different organisms.

Thinking Process

Gel electrophoresis, developed by A Tiselws is used in separation of molecule like protein, DNA and RNA. Agarose is commonly used matrix (polysaccharides) in this technique (polysaccharides).

Answer

(b) In agarose gel electrophoresis, the DNA fragments separate out (resolve) according to their size or length because of the sieving property of agarose gel. It means, the smaller the fragment size, the farther it will move.

Answer

(a) All of the given features are important to facilitate cloning into a vector but out of them Origin of replication (ori) is the most important one.

This is due to the following reasons

(i) Ori is a DNA sequence that is responsible for initiating replication. Any piece of DNA when linked to this sequence can replicate within the host cells.

(ii) Ori also controls the copy numbers of the linked DNA.

Diagram showing essential features of plasmid pBR322

Answer

(c) In the process of ‘recombinant DNA technology’ the first step is isolation of DNA. Since, the DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids.

This can be achieved by treating the bacterial cells/plant of animal tissue with enzymes such as lysozyme (bacteria), cellulase (plant cells) and chitinase (fungus).

As we know that genes are located on long molecules of DNA interwined with proteins such as histones. The RNA can be removed by treatment with ribonuclease, whereas proteins can be removed by treatment with protease.

Other molecules can be removed by appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol. Deoxyribonuclease is not used in this process as this enzyme causes the lysis of DNA molecules.

Answer

(d) The Polymerase Chain Reaction (PCR) is a reaction in which amplification of specific DNA sequences is carried out in vitro. Such repeated amplification is achieved by the use of a thermostable DNA polymerase (isolated from a bacterium, Thermus aquaticus), which remain active and stable during the high temperature and induced denaturation of double-standard DNA.

Thinking Process

The genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc., are considered as useful selectable markers for E. coli.

Answer

(b) Selectable markers help in identifying and eliminating non-transformants and selectively permitting the growth of the transformants. The normal $E$. coli cells do not carry resistance against any of these antibiotics. Competant bacterial cells are made capable to take foreign DNA with chemical treatment, e.g., calcium chloride.

Note In process of transformation, a piece of DNA is introduced in a host bacterium.

Thinking Process

DNA being a hydrophilic molecule cannot pass through cell membranes. Therefore, the bacteria should be made competent to accept the DNA molecules by chemical and physical methods.

Answer

(c) In chemical method, the cell is treated with specific concentration of a divalent cation such as calcium to increase pore size in cell wall. The cells are incubated with recombinant DNA on ice, followed by placing them briefly at $42^{\circ} \mathrm{C}$ and then putting it back on ice. This is called heat shock method. The bacteria now takes up the recombinant DNA.

Answer

(a) DNA ligase (joining or sealing enzymes) are also called genetic gum. They join two individual fragments of double-stranded DNA by forming phosphodiester bonds between them. Thus they help in sealing gaps in DNA fragments. Therefore, they act as a molecular glue.

Answer

(c) Agrobacterium tumefaciens is a pathogen of several dicot plants. It delivers a piece of DNA known as ‘T-DNA’ in the Ti plasmid which transforms normal plant cells into tumour cells to produce chemicals against pathogens.

The restriction enzyme Eco RI, is isolated from Escherichia coli RY13.

The first restriction enzymes Hind II was isolated from bacterium Haemophilus influenzae. The restriction enzyme Bam HI is isolated from Bacillus amyloli.

Answer

(c) In polymerase chain reaction polymerisation or extension step is catalysed by Taq polymerase enzyme. PCR is carried out in the following three steps

(i) Denaturation The double-stranded DNA is denatured by applying high temperature of $95^{\circ} \mathrm{C}$ for 15 seconds. Each separated single stranded strand now acts as template for DNA synthesis.

(ii) Annealing Two sets of primers are added which anneal to the 3 end of each separated strand. Primers act as initiators of replication.

(iii) Extension DNA polymerase extends the primers by adding nucleotides complementary to the template provided in the reaction.

A thermostable DNA polymerase (Taq DNA polymerase) is used in the reaction which can tolerate the high temperature of the reaction.

All these steps are repeated many times to obtain several copies of desired DNA.

Thinking Process

Introns are parts of genes that do not directly code for proteins. There are commonly found in multicellular eukaryotes, such as humans and rare in bacteria.

Answer

(a) The process of making recombinant DNA molecule involves the introduction of a desired gene into the DNA of a host that will produce the desired protein.

Inducing a cloned eukaryotic gene to function in a prokaryotic host can be difficult sometime. The presence of long non-coding introns in eukaryotic genes may prevent correct expression of these genes in prokaryotes, which lack RNA-splicing machinery.

Answer

(c) If any protein encoding gene is expressed in a heterologous host. it is called a recombinant protein. The cells harbouring cloned genes of interest may be grown on a small scale in the laboratory.

The cultures may be used for extracting the desired protein and then purifying it by using different separation techniques.

The cells can also be multiplied in a continuous culture system where in the used medium is drained out from one side while fresh medium is added from the other to maintain the cells in their physiologically most active log/exponential phase. This type of culturing method produces a larger biomass leading to higher yields of desired protein.

Answer

(c) PCR (Polymerase Chain Reaction) technique was developed by Kary Mullis in 1985, and for this he received Nobel Prize for chemistry in 1993. HG Khurana discovered DNA ligase enzyme in to phage in 1969.

White DNA polymerase was discovered by Arthur Kornberg and Herbert Boyer generated first recombinant DNA molecule by combining a gene from a bacterium with plasmid of $E$. coli in 1972.

PCR (Polymerase Chain Reaction) technique is a reaction in which amplification of specific DNA sequences is carried out in vitro.

Answer

(c) The restriction enzymes are called ‘molecular scissors’ and are responsible for cutting DNA on specific sites. These are not found in viruses.

They are present in bacteria to provide a type of defense mechanism called the ‘restriction modification system’ and the so called system consist of two component; restriction enzymes and modification enzyme.

The first component include restriction endonuclease, which identify the introduced foreign DNA and cut it into process. Same kind of sticky end in different individual molecule of DNA are also produced by these molecular scissors special sequence in the DNA recognised by restriction endonuclease is called palindromic nucleotide sequence.

Answer

The recombinant DNA can multiply as many times as the copy number of the vector plasmid thereby determining the yield of recombinant protein. So, higher the copy number of plasmid vector, higher the copy number of gene and consequently, protein coded by the gene is produced in high amount.

Answer

No, as exonuclease acts on the free ends of linear DNA molecule. Therefore, instead of producing DNA fragments with sticky ends, it will shorten or completely degrade the DNA fragment containing the gene of interest and the circular plasmid (vector) will not get cut as it lacks free ends.

Answer

(i) The first letter ’ $H$ ’ indicates the genus of the organism from which the enzyme was isolated, $H=$ genus Haemophilus.

(ii) The fourth letter $d$ indicates the particular strain used to produce the enzyme, $d=$ strain Rd.

(iii) The Roman numerals denoted the sequence in which the restriction endonuclease enzyme from that particular genus, species and strain of bacteria have been isolated-III, i.e., third restriction endonuclease to be isolated from this species.

Answer

If the restriction enzymes have more than one recognition site in a vector, then the vector itself will get fragmented on treatment with the restriction enzymes.

Answer

DNA being a hydrophilic molecule can not pass through cell membranes. Therefore, the bacteria should be made competent to accept the DNA molecules.

Competent means bacterial cells, on treatment with chemicals like $\mathrm{CaCl} _{2}$, are made capable of taking up foreign DNA.

Answer

Proteases degrade the proteins present inside a cell (from which DNA is being isolated). If the proteins are not removed from DNA preparation then they could interfere with any downstream treatment of DNA.

Answer

If denaturation of double-stranded DNA does not take place then primers will not be able to anneal (joining) to the template. Hence, no extension will take place and after are there will be no amplification.

Answer

Hepatitis-B recombinant vaccine (engerix) is used for vaccination of hapatitis virus.

Thinking Process

Water is critical not only for the correct folding of proteins but also for the maintenance of the structure of DNA and protein.

Answer

Biomolecules (DNA, and protein) exhibit change in biological activity in anhydrous conditions, In non-aqueous or anhydrous conditions the rigidity of protein and DNA increases due to the weakening of hydrogen bond strength.

It results into the change in overall free energy, which is the combined effects of the exposure of the interior polar and non-polar groups and their interaction with water In absence of aqueous condition, the free energy change is negative, which is responsible for the denaturation of biomolecules.

Increasing strength of hydrogen-bond causes water to primarily bond with itself and not to be available for the hydrating structure of proteins or DNA, or for dissolving ions.

On the other hand, if the water-water hydrogen bond strength reduces then the exchange mechanisms operating within the cell, such as hydrogen bonded water chains within and between proteins and DNA, will become non-operational. It will further leads to the denaturation

Thinking Process

T-DNA is the only essential part required to make Ti-plasmid a cloning vector.

Answer

The plasmid is disarmed by deleting the tumour inducing genes in the plasmid. So, that it become an effective cloning vector. The modified tumour inducing (Ti) plasmid of Agrobacterium tumefaciens will no longer remain pathogenic to the plants but still deliver genes of interest into a variety of plants.

Answer

Gene cloning refers to a process in which a gene of interest is ligated to a vector. The recombinant DNA thus produced is introduced in a host cell by transformation.

Each cell gets one DNA molecule and when the transformed cell grows to a bacterial colony, each cell in the colony has a copy of the gene. This is gene cloning.

Answer

In my opinion both of them are correct. As biotechnology is a very wide area which deals with techniques of using a ’natural’ organism (or its parts) as well as genetically modified organism to produce and processes useful for mankind.

A wine maker employs a strain of yeast to produce wine by fermentation (a natural phenomenon), while the molecular biologist has cloned gene for the antigen (that is used as vaccine) in an organism which allows the production of the antigen in large amount.

Thinking Process

Bacterial transformation is the process by which bacterial cells take up naked DNA molecules (exogenous or foreign DNA).

Answer

The experiment will not likely to be affected as recombinant DNA molecule is circular and closed, with no free ends. Hence, it will not be a substrate for exonuclease enzyme which removes nucleotides from the free ends of DNA.

Answer

If the restriction enzymes would cut DNA at random sites instead of at specific sites, then the DNA fragments obtained will not have ‘sticky ends’. In the absence of sticky ends, construction of recombinant DNA molecule would not be possible.

Answer

When a plasmid DNA and a linear DNA having one site for a restriction endonuclease are cut and separated, plasmid shows one DNA band, while linear DNA shows two band because of difference in their basic structure.

Plasmid is a circular DNA molecule and when cut with these enzyme, it becomes linear but does not get fragmented due to presence of only one restriction site, whereas a linear DNA molecule gets cut into two fragment.

Answer

A compound called ethidium bromide stains DNA, which on exposure with ultra-violet, (uv) radiation gives orange light band of DNA. Hence, DNA fragments appear as orange band in the presence of ethidium bromide and UV light.

Answer

In a gene cloning experiment, first a recombinant DNA molecule is constructed, where the gene of interest is ligated to the vector (the step would not be affected) and introduced inside the host cell (transformation)

Since, not all the cells get transformed with the recombinant/plasmid DNA, in the absence of selectable marker, it will be difficult to distinguish between transformants and non-transformant, because role of selectable marker is in the selection of transformants.

Answer

The reasons are as follows

(i) DNA sample that was loaded on the gel may have got contaminated with nuclease (exo or endo both) and completely degraded.

(ii) Electrodes were put in opposite orientation in the gel assembly that is anode towards the wells (where DNA sample is loaded). Since, DNA molecules are negatively charged, they move towards anode and hence, move out of the gel instead of moving into the matrix of gel.

(iii) Ethidium bromide was not added at all or was not added in sufficient concentration and so DNA was not visible.

Answer

$\mathrm{CaCl}_{2}$ is known to increase the efficiency of DNA uptake to produce transformed bacterial cells. The divalent $\mathrm{Ca} ^{+2}$ ions create transient pores on the bacterial cell wall by which the entry of foreign DNA is facilitated into the bacterial cells.

Answer

In the absence of antibiotic, there will be no pressure on recombinants to retain the plasmid (containing the gene of our interest). Since, maintaining a high copy number of plasmids is a metabolic burden to the microbial cells, it will thus tend to loose the plasmid.

Answer

Region to be amplified

In PCR, each cycle has three steps

(i) Denaturation of DNA Sample Unwinding of two strand of DNA by heating the sample at $92-94^{\circ} \mathrm{C}$.

(ii) Primer Annealing Primers get positioned on the exposed nucleotides as per base pairing rules.

(iii) Extension of Primers DNA polymerase recognises primers as ‘start’ tags and begins to extend the primers using the free nucleotides provided in the reaction and the genomic DNA as template.

With each round of reactions, the DNA doubles.

Answer

Region A Bam $\mathrm{HI}$

Region $B$ Pot I

Region $\mathrm{C}$ amp $^{R}$.

E. coli cloning vector pBR322 showing restriction sites (Hind III, Eco RI, Bam HI, Sal I, Pvu II, Pst I, Cla I), Ori and antibiotic resistance genes $(\mathrm{amp} ^{\mathrm{R}}$ and $\mathrm{tet}^{\mathrm{R}})$.

Rop codes for the proteins involved in the replication of the plasmid.

Answer

In selection of recombinants due to inactivation of antibiotics, the transformed cells are first plated on the antibiotic plate which has not been insertionally inactivated (i.e., ampicillin) and incubated overnight for growth of transformants.

For selection of recombinants, these transformants are replica-plated on second antibiotic (say, tetracycline) plate (which got inactivated due to insertion of gene).

Non-recombinants grow on both the plates (one carrying ampicillin and the other carrying tetracycline) while recombinants will grow only on ampicillin plate. This entire exercise is labourious and takes more time (two overnight incubation) as well.

However, if we choose insertional inactivation of a marker that produces colour in the presence of a chromogenic compound, we can distinguish between the recombinants and non-recombinants on a single medium plate (containing one antibiotic and the chromogenic compound) after overnight growth.

Answer

A soil-inhabiting, plant pathogenic bacterium, Agrobacterium tumefaciens, infects broad-leaved crops including tomato, soyabean, sunflower and cotton, but not the cereals. It causes tumours called crown galls.

Tumour formation is induced by its plasmid, which is, therefore called Ti-plasmid (Ti for tumour inducing). The Ti-plasmid integrates a segment of its DNA, termed T-DNA, into the chromosomal DNA of its host plant cells. The T-DNA causes tumours. As gene transfer occurs without human effort, the bacterium is known as natural genetic engineer of plants.

Plant molecular biologists have started using Ti-plasmids as vectors to transfer foreign genes of interest into the target plant cells. They use a version of the plasmid from which tumour forming gene has been eliminated. The transformed bacteria do not cause disease, but still deliver genes of interest into a variety of plants.

Answer

Bioreactors are vessels of large volumes (100-1000 L) in which raw materials are biologically converted into specific products.

The most commonly used bioreactors are of stirring type, which are shown in figure.

(a)

(b)

(a) Simple stirred-tank bioreactor

(b) Sparged stirred-tank bioreactor through which sterile air bubbles are sparged

A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. Alternatively air can be bubbled through the reactor.

If you look at the figure closely you will see that the bioreactor has an agitator system, an oxygen delivery system and a form control system, a temperature control system, $\mathrm{pH}$ control system and sampling ports so, that small volumes of the culture can be withdrawn periodically.

Small volume cultures are usually employed in laboratories in a flask for research and production of less quantities of products. However, large scale production of the products is carried out in bioreactors.