Exercise
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).
Sol. Recombinant proteins are the proteins that are produced through recombinant DNA technology. These proteins are produced from the expression of protein encoding gene in another organism after its transfer using vectors. 10 recombinant proteins used in medical practices and their therapeutic uses are as follows:
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.
Sol. Restriction enzymes are endonucleases which recognise a specific palindromic nucleotide sequences in the DNA (called recognition sites) and make cuts in each of the two strands of the double helix at specific points within their recognition sites. For example, BamHl is a restriction enzyme whose recognition site is 6 base pairs in length.
The following chart showing the substrate DNA, the cleavage site and the restriction products of BamHl is given below.
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?
Sol.
- Though both DNA and enzymes are macromolecules, DNA is bigger in molecular size as compare to enzyme.
- This is because DNA is a polymer of deoxynucleotides and contains the information for the synthesis of proteins. In comparison, enzymes are proteins which are polymers of amino acids of variable number synthesised from a small segment of DNA called gene.
Question 4. What would be the molar concentration of human DNA in a human cell? Consult your teacher.
Sol. The molar concentration of DNA is the molar concentration of the total nucleotides present in one human cell.
Genomic DNA per human cell= 6 picogram = 6 x 10-12 g
Average molar mass of a nucleotide= 327g/mol Number of nucleotides in human genome= 6.6 x 109
Question 5. Do eukaryotic cells have restriction endonucleases? Justify your answer.
Sol.
- No, eukaryotic cells do not have restriction endonucleases.
- All the restriction endonucleases have been isolated from various strains of bacteria (prokaryotes). They possess these enzymes as a defence mechanism to destroy the foreign DNA or to restrict the growth of invading viruses called bacteriophages. Such mechanism to restrict the growth of invading viruses is absent in eukaryotes.
Note: DNA of eukaryotes is highly methylated by a modification enzyme, called methylase. Methylation protects the DNA from the activity of restriction enzymes.
Question 6. Besides better aeration and mixing properties, what other advantages do stirred tank bioreactors have over shake flasks?
Sol.
- Shake flasks are used for growing and mixing the desired materials on a small scale in the laboratory. A large scale production of desired biotechnological products (100-1000 litres) is done by using ‘bioreactors’. The most commonly used bioreactors are of stirring type. 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.
- Besides better aeration and mixing properties, stirred tank bioreactors have following advantages:
- It has sampling parts from where small volumes of cultures can be periodically withdrawn from the reactor for sampling.
- It has a foam control system, pH control system and temperature control system.
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.
Sol. A palindromic DNA sequence is a sequence of base pairs that reads the same on the two strands when the orientation of reading of sequences in DNA is kept the same, i.e., from 5′ 3′ or 3′ 5′.
Question 8.Can you recall meiosis and indicate at what stage a recombinant DNA is made?
Hint: Third stage of Meiosis 1, that is characterised by paired chromosomes thickened and visibly divided into chromatids and by the occurrence of crossing over.
Sol. Recombinant DNA is the DNA with new combination formed by the joining of two or more gene sequences. It is made during pachytene stage of prophase I of meiosis I. This is the stage when crossing over involving exchange of DNA segments between non-sister chromatids of homologous chromosomes takes place.
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?
Sol. Transformation is a procedure through which a piece of DNA is introduced in a host bacterium. Selectable markers are the genes present in vector that help in identification and elimination of non-transformants by selectively permitting the growth of the transformants. Genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc., are examples of selectable markers.
Selection of recombinants due to inactivation of antibiotics is a cumbersome procedure because it requires simultaneous plating on two plates having different antibiotics. So gene encoding reporter enzyme linked to the gene of interest is used to monitor transformation of host cells. Such vectors have a reporter enzyme in addition to antibiotic resistance gene and recognition sites for restriction enzymes are present in gene encoding reporter enzyme. Reporter enzymes convert invisible substrates to luminescent or coloured products. Examples of reporter enzymes are p-galactosidase or alkaline phosphatase. If the bacteria transformed with vector without an insert, are grown on the medium containing substrate of p-galactosidase, blue coloured colonies are produced. If bacteria transformed with an insert within the coding sequence of p-galactosidase are grown on the medium containing substrate, the colonies do not produce any colour and form white colonies due to insertional inactivation of p-galactosidase. These white colonies are identified as recombinant colonies. Thus, on the basis of colour of colony, transformed host cells are selected.
Note: Use diagrammatic representation to understand the insertional inactivation of enzyme which is responsible for a coloured product. The visual will help in recall
Question 10. Describe briefly the followings:
(a) Origin of replication (b) Bioreactors (c) Downstream processing
Sol. (a) Origin of replication (Ori)
Ori is a specific DNA sequence from where replication initiates and any piece of DNA when linked to this sequence can be made to replicate within the host cells. This sequence is also responsible for controlling the copy number of the linked DNA. So, if one wants to recover many copies of the target DNA it should be cloned in a vector whose origin support high copy number.
(b) Bioreactors
Bioreactors are vessels in which raw materials are biologically converted into specific products by microbes, plant and animal cell and/or their enzymes. The bioreactor provides optimum growth conditions for achieving the desired products. The most commonly used bioreactor is of stirring type. An astirred tank bioreactor is usually a cylindrical vessel or vessel with a curved base to facilitate mixing of the contents. In the sparged stirred tank bioreactor, sterile air bubbles are sparged. The stirrer facilitates the mixing and oxygen availability throughout the bioreactor. A bioreactor has an agitator system, an oxygen delivery system, a foam control system, a temperature control system, pH control system and sampling ports.
(c) Downstream processing
The product once obtained is subjected to a series of processes collectively called downstream processing before it is made into a finished product ready for marketing. The two main processes are separation and purification. The product is then formulated with suitable preservatives. Such formulations have to undergo clinical trials, in case of drugs. The downstream process and quality control test are different from product to product.
Question 11. Explain briefly
- (a) PCR
- (b) Restriction enzymes and DNA
- (c) Chitinase
Sol. (a) PCR
- PCR or Polymerase chain reaction is a technique (developed by Kary Mullis in l 985) to synthesise multiple copies of gene or DNA of interest using a set of primers and DNA polymerase in vitro.
- Thermostable DNA polymerase, Taq DNA polymerase isolated from a bacterium, Thermus aquaticus is used in PCR. This enzyme remains active during the high temperature induced for the denaturation of double-stranded DNA.
- The process of amplification of gene of interest using PCR technique involves following three steps:
- Denaturation: In this step, reaction mixture heated to separate double-stranded template DNA into two single strands. Annealing : In this step, the reaction mixture is cooled so the primers can bind to their complementary sequences on the single-stranded template DNA strands.
- Extension: In this step, temperature of the reaction mixture is raised to 72°C so Taq polymerase extends the primers using the nucleotides provided in the reaction and the genomic DNA as template, synthesising new strands of DNA.
These steps are repeated in a cyclic manner and the gene of interest is amplified to approximately billion times, i.e., 1 billion copies are made.
Note:
- PCR is based on the principle that a DNA molecule, when subjected to high temperature, splits into two strands due to denaturation.
- Applications of PCR: diagnosis of pathogens, diagnosis of specific mutations, DNA fingerprinting, in prenatal diagnosis and gene therapy.
(b) Restriction enzymes and DNA
- Restriction enzymes are a class of nucleases present in bacteria responsible for restricting the growth of bacteriophages.
- They cut DNA molecules at a particular point by recognising a specific palindromic sequence of four to eight base pairs known as the recognition sequence. Once it finds its specific recognition sequence, it will bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar-phosphate backbones.
- Restriction endonucleases are used in genetic engineering to form recombinant DNA molecules from different sources or genomes. When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of sticky-ends and, these can be joined together (end-to-end) using DNA ligases. Thus, these are called molecular scissors.
Note: Hind11wasthefirstrestrictionendonucleasetobediscovered.Hind 11always cuts DNA molecules at a particular point by recognising a specific sequence of six base pairs. This specific base sequence is called the recognition sequence for Hind 11.
(c) Chitinase
- It is an enzyme used during DNA isolation from fungal cells.
- This enzyme acts on the chitin present in the cell wall of fungal cells and breaks them open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids.
Question 12. Discuss with your teacher and find out how to distinguish between
- (a) Plasmid DNA and Chromosomal DNA
- (b) RNA and DNA
- (c) Exonuclease and Endonuclease
Sol. (a) Difference between plasmid DNA and chromosomal DNA
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