KENDRYA VIDYALAYA NO.

7 CISF
BIOTECHNOLOGY: PRINCIPLES AND PROCESS
1. Nihal said that bacteria are never used as a host to clone recombinant DNA for antibiotic production. Is
his statement correct and why?

A. Yes, because antibiotics kill host bacterial cells.

B. No, because antibiotics only kill cells other than bacterial cells.

C. No, because the foreign DNA or the host bacterial cell is resistant to the antibiotic being produced.

D. Yes, because host bacterial cells do not have the machinery to allow the growth of an antibiotic
obtained from other microbes.

2. A researcher used a vector X to insert a foreign gene to create a recombinant vector. The image of
vector X is shown below.

It has sites for two restriction enzymes - SacI and EcoRI. The foreign gene can be cut using either of
these two enzymes. The vector also has a green fluorescent protein (gfp) gene that can be used as a
selectable marker, and two genes - chloramphenicol resistance (CmR) and neomycin resistance
(NeoR) that provide antibiotic resistance. Chloramphenicol and neomycin are two different antibiotics.

(a) What is/are the possible end product(s) that will be obtained post-ligation if the researcher uses the
following enzyme to insert the foreign gene:

(i) SacI

(ii) EcoRI

(b) Based on (a), which enzyme will be better to use to ensure that the foreign gene has been inserted
in the vector? Why?

(c) If the well of an agarose gel is filled with a solution of the intact vector and the foreign gene, what
will the DNA band closer to the well contain? Why?

3. Bacterial cells offer certain advantages over plant or animal cells that make them an easy choice for
the production of many recombinant molecules. State THREE such advantages.
4. Give TWO reasons why it is important to introduce the gene/s of interest in a vector and then into the
host cell or insert it directly into the host chromosomal genome.
5. The large-scale production of an organism is generally done in a bio-processor unit. Given below is the
growth curve of a bacteria that is being used for the production of a recombinant molecule. Maintaining
sterile conditions is of utmost importance in a bio-processor unit.
 (a) In which phase are the cells likely to be producing a larger concentration of the recombinant
molecule? Why?

(b) In cases where the culture in the bio-processor unit reaches the death phase, identify ONE strategy
that can help revive the bio-processing to restart production of the recombinant molecule.

(c) What does a sterile condition mean?

(d) State ONE reason why the bacteria that are producing the recombinant molecule are not harmed
during the process of sterilisation.

6. pBR322 was a plasmid that was constructed artificially using genetic material from three sources:

i) the tetracycline resistance gene of pSC101

ii) the ampicillin resistance gene of RSF 2124

iii) the replication elements of pMB1, a close relative of the ColE1 plasmid.

Describe the TWO enzymatic steps that would have occurred in the construction of pBR322.

7. In the process of DNA replication, RNA primers are used to initiate replication where a free nucleotide
can start forming a bond with the RNA primer as per the leading strand base sequence. Later, this RNA
primer needs to be removed. What type of a nuclease can help with this activity? Why?
8. The basis of rDNA technology is to alter the genetic material of an organism to obtain enhanced and
desired characteristics in an organism.

(a) Preferably, the gene of interest and the vector are cut with the same restriction enzyme. Is this
statement true? Give a reason to support your answer.

(b) What is/are ALL the possible outcomes after the gene of interest and the vectors are ligated?

(c) If a vector contains an ampicillin resistance gene as the selectable marker, state TWO situations in
which the host cell will grow in a medium that contains ampicillin.

9. State whether each of these statements given below is/are true or false. Justify your answer.

(a) Plasmids with a single restriction site are preferred over those with multiple sites for the same
enzyme during the cloning process.

(b) The tumour-inducing (Ti) plasmid can be extracted from Agrobacterium tumifaciens cells and used
as it is for cloning a foreign gene.

10. Vectors containing the foreign DNA have to be forced into host cells that are made competent to do so.
A common method is to first treat host cells with calcium chloride and then incubate these cells on ice.
This is followed by briefly placing them at 42 °C and then putting them back on ice. This enables the
host cells to take up recombinant DNA and is called the heat shock treatment.
 (a) Explain why DNA vectors CANNOT pass through the cell membrane like other molecules such as
oxygen.

(b) Calcium from a CaCl2 solution binds to DNA making it easier for them to enter a cell. Why?

(c) Why does the heat shock treatment make the membrane porous allowing for easy uptake of DNA?

11. Polymerase chain reaction (PCR) is an in-vitro technique used to amplify nucleic acid sequences. The
conditions and duration of each step in PCR are as follows:

- Step 1 at 94 °C for 2 min

- Step 2 at 50-65 °C for 30 seconds

- Step 3 at 72 °C for 5 min

(a) Give TWO reasons why amplification using PCR can be better than amplification in-vivo using
plasmids.

(b) At which step does the denaturation of DNA take place? How does this occur?

(c) What would be the result of the PCR reaction if step 2 does not occur?

(d) At what step would PCR be important in rDNA technology?

12. Insulin is commonly prepared in bio-processing units for patients suffering from insulin-dependent
diabetes. Explain THREE steps that would be a part of the downstream processing for insulin.
13. Which of the following is a restriction enzyme?

a) DNA polymerase b) RNA polymerase

c) EcoRI d) Ligase

14. The process of transferring DNA into bacterial cells is called:

a) Transcription b) Translation

c) Transformation d) Translocation

15. Which enzyme is used to join DNA fragments?

a) DNA polymerase b) DNA ligase

c) RNA polymerase d) Restriction enzyme

16. What does PCR stand for?

a) Protein Chain Reaction b) Polymerase Chain Reaction

c) Plasmid Chain Reaction d) Polypeptide Chain Reaction

17. Which of the following is a commonly used vector in genetic engineering?

a) RNA b) Plasmid

c) Ribosome d) Golgi apparatus

18. In gel electrophoresis, DNA fragments are separated based on:

a) Size b) Charge

c) Shape d) Colour
19. Taq polymerase is obtained from:

a) Escherichia coli b) Saccharomyces cerevisiae

c) Thermus aquaticus d) Bacillus subtilis

20. The term 'recombinant DNA' refers to:

a) RNA molecules joined together b) DNA molecules from two different sources

c) DNA and RNA combined d) Proteins synthesized from DNA

21. Which of the following is used as a selectable marker in cloning vectors?

a) Antibiotic resistance gene b) LacZ gene

c) Ori site d) None of the above

22. The technique used to amplify DNA is:

a) Southern blotting b) Northern blotting

c) PCR d) Gel electrophoresis

23. What is the role of the origin of replication in a plasmid?

a) Initiates DNA replication b) Terminates DNA replication

c) Degrades DNA d) Modifies DNA

24. Which of the following is an example of a bioreactor?

a) Test tube b) Petri dish

c) Fermenter d) Microscope

25. The enzyme used to cut DNA at specific sites is:

a) DNA ligase b) RNA polymerase

c) Restriction enzyme d) DNA polymerase

26. Which of the following is used to visualize DNA fragments after gel electrophoresis?

a) Ethidium bromide b) Coomassie blue

c) Silver stain d) Bromophenol blue

27. The process of inserting foreign DNA into a host cell is known as:

a) Transcription b) Translation

c) Transformation d) Translocation