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Q1. What are recombinant proteins? How do bioreactors help in their production?

Solution

The protein produced by genetically altered DNA (recombinant DNA) in a heterologous host is called recombinant protein. Bioreactors are vessels in which raw materials are biologically converted into specific products by microbes. It provides optimum growth conditions such as temperature, pH, substrate, vitamins, oxygen and salts for the production of recombinant proteins.

Q2. For selection of recombinants, insertional inactivation of antibiotic marker has been superceded by insertional inactivation of a marker gene coding for a chromogenic substrate. Give reasons.

Solution

Selection of recombinants due to inactivation of antibiotic coding gene is a laborious process as it requires: (i) a vector with two antibiotic resistance markers (ii) preparation of two kinds of media plates, each with one type of antibiotic. Transformed cells are first plated on the antibiotic plate in which the antibiotic gene has not been insertionally inactivated (example: ampicillin) and incubated overnight for the growth of transformants. For selection of recombinants, these transformants are replica-plated on the plate containing second antibiotic plate (example, tetracycline) which got inactivated due to insertion of the 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 process is laborious and requires more time (two overnight incubation) as well. However, if we choose insertional inactivation of a marker that produces colour in presence of a chromogenic substance, we can easily distinguish between recombinants and non-recombinants on a single medium plate (containing one antibiotic and the chromogenic compound) after just overnight growth.

Q3. What is Ti plasmid? Name the organism where it is found. How does it help in genetic engineering?

Solution

An extra-chromosomal DNA which delivers gene of interest into a variety of plants and acts as cloning vector in the host organism is called Ti plasmid. It is present in pathogenic bacterium Agrobacterium tumefaciens. Ti plasmid vectors are used for genetic transformation in many dicot plants. The tumour inducing (Ti) plasmid of Agrobacterium tumefaciens has been modified into a cloning vector which is no more pathogenic to the plants, but is still able to use the mechanism to deliver the gene of interest into a variety of plants.

Q4. How can DNA segments be separated by gel electrophoresis, visualised and isolated?

Solution

As pure DNA fragments cannot be seen under visible light, they can be visualised only after staining them with a solution of ethidium bromide followed by exposure to UV radiation. They appear as bright orange coloured bands. The separated bands of DNA are then cut from the agarose gel and extracted by using a convenient technique. This process is called elution. The eluted DNA fragments are then purified and used in constructing recombinant DNA by joining them with cloning vectors.

Q5. Explain any two methods of vectorless gene transfer.

Solution

The two methods of vectorless gene transfer are as shown below: (i) Microinjection: The technique of introducing foreign DNA into a target cell by injecting the DNA directly into the nucleus with the help of a micro-needle is called micro-injection. (ii) Electroporation: The process in which transient holes are produced in the plasma membrane of the target cell to incorporate foreign DNA is called electroporation.

Q6. Who developed the technique of electrophoresis? State its principle.

Solution

Electrophoresis was developed by Tiselius in 1937 as a method for the separation of substances with different ionic properties. It is based on the principle that charged particles move under the influence of electric current to oppositely charged electrodes.

Q7. How is the amplification of a gene sample of interest carried out using Polymerase Chain Reaction (PCR)?

Solution

Polymerase Chain Reaction (PCR) is a technique used to synthesize multiple copies of the desired gene in vitro within a short span of time. Amplification of a gene sample of interest is carried out using PCR in the following three steps: (a) Denaturation: The double stranded DNA is denatured by applying high temperature of about 95C for 15 seconds. Each separated single stranded strand now acts as template for the synthesis of new DNA strand. (b) Annealing: Two sets of primers are added which anneal at the 3’ end of each separated strand. They mark the beginning of replication. (c) Extension: A thermostable DNA polymerase, Taq polymerase extends the primers by adding nucleotides complementary to the template provided in the reaction. The cycle of denaturation, annealing and extension is repeated several times to obtain several copies of desired DNA.        

Q8. (i) Mention the number of primers required in each cycle of polymerase chain reaction (PCR). Write the role of primers and DNA polymerase in PCR. (ii) Give the characteristic feature and source organism of DNA polymerase used in PCR.

Solution

(i) Each cycle of PCR requires two sets of primers. Role of primers: Primers are complementary to the regions of DNA. They anneal to both the strands of DNA which are then extended using nucleotides. Role of DNA polymerase: The enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as template. (ii) DNA polymerase used in PCR is thermostable and active even at a temperature of 72C and is called Taq polymerase. It is obtained from the bacterium Thermus aquaticus.

Q9. What is a cloning vector? Explain the technique of using such a vector in E.coli.

Solution

Cloning vectors are DNA molecules used as carriers for transferring a fragment of foreign DNA and capable of replicating inside the host cell independent of the control of chromosomal DNA. Plasmids and bacteriophages are commonly used cloning vectors. The technique of using cloning vector in E. coli is as follows: (i) The foreign DNA is linked to the sequence called ori (origin of replication) and is made to replicate within the host cell. (ii) The ligation of foreign DNA is carried out at a restriction site present in the resistance genes. (iii) The foreign DNA is then attached to the plasmid DNA (vector) with the help of enzyme ligase. (iv) This recombinant DNA (rDNA) is inserted into the bacterial cell where it multiplies to produce multiple copies of the desired gene.        

Q10. (i) Describe the characteristics that a cloning vector must possess. (ii) Why DNA cannot pass through the cell membrane? Explain. How is a bacterial cell made 'competent' to take up recombinant DNA from the medium?

Solution

(i) A cloning vector must possess the following characteristics: (a) Relaxed origin of replication (ori) (b) Selectable marker, genes encoding for an antibiotic resistance or genes encoding for -galactosidase to identify and eliminate non-transformants. (c) Cloning site or recognition site for the restriction enzyme to recognise. (ii) DNA is a hydrophilic molecule. Therefore, it cannot pass through the cell membrane. The bacterial cells can be made competent by treating them with a specific concentration of a divalent ion like calcium. The cells are then incubated on ice followed by a heat shock treatment by placing them briefly at 42C and then putting back on ice.