Biotechnology: Principles and Processes

Chapter 9: Biotechnology - Principles and Processes

Multiple Choice Questions

Genetic Engineering Roles
- Which of the following does not have a direct role in genetic engineering?
  - (a) Restriction enzymes
  - (b) Polymerase
  - (c) Lipase
  - (d) Vectors
- Correct Answer: (c) Lipase
Artificial Recombinant DNA Molecule
- The first instance of construction of an artificial recombinant DNA molecule was carried out on the plasmid of:
  - (a) Bacillus thuringiensis
  - (b) Escherichia coli
  - (c) Agrobacterium tumefaciens
  - (d) Salmonella typhimurium
- Correct Answer: (b) Escherichia coli
Cohen and Boyer
- Cohen and Boyer created the first recombinant DNA using plasmid of:
  - (a) Escherichia coli
  - (b) Thermus aquaticus
  - (c) Salmonella typhimurium
  - (d) Bacillus thuringiensis
- Correct Answer: (a) Escherichia coli
Gel Electrophoresis Visualization
- In gel electrophoresis, the separated DNA fragments can be visualized:
  - (a) In visible light.
  - (b) In visible light with staining.
  - (c) In UV radiation without staining.
  - (d) In UV radiation after staining with etherium bromide.
- Correct Answer: (d) In UV radiation after staining with ethidium bromide.
Competent Cells
- Which of the following is used to make the bacterial cell a ‘Competent cell’ to take up DNA?
  - (a) Carbonate ion
  - (b) Calcium ion
  - (c) Sodium ion
  - (d) Sulphate ion
- Correct Answer: (b) Calcium ion
Enzymes in Gene Cloning
- Which of the following enzyme(s) are NOT essential for gene cloning?
  - A. Restriction enzymes
  - B. DNA ligase
  - C. DNA mutase
  - D. DNA recombinase
  - E. DNA polymerase
- Correct Answer: C. DNA mutase

Assertion-Reason Questions

Assertion A: Polymerase chain reaction is used in DNA amplification.
Reason R: The ampicillin resistant gene is used as a selectable marker to check transformation.
Answer Options:
- (a) Both correct, R explains A.
- (b) Both correct, R does not explain A.
- (c) A correct but R wrong.
- (d) A wrong but R correct.
  - Correct Answer: (c)
Assertion A: When a particular restriction enzyme cuts a strand of DNA, overhanging stretches or sticky ends are formed.
Reason R: Some restriction enzymes cut the strand of DNA a little away from the center of the palindromic site.
- Correct Answer: (a)
Assertion A: In GMOs desirable DNA segment is introduced into a suitable host.
Reason R: The gene transfer is done using a biolistic gun only.
- Correct Answer: (c)
Assertion A: The palindromic sequences at which endonucleases act vary from organism to organism.
Reason R: The palindromic sequence of Eco RI is GAATTC.
- Correct Answer: (b)
Assertion A: A temperature control system is an important requirement for a bioreactor.
Reason R: Every microorganism or enzyme is functional only at optimum temperature conditions.
- Correct Answer: (a)

Short Answer Questions

Difference between Exonuclease and Endonuclease
- Exonucleases remove nucleotides from the ends of the DNA molecule, whereas endonucleases cut the DNA strand at specific points within the strand.
Host for Foreign Gene Product
- The host that produces a foreign gene product is called a host organism. The product itself is known as a recombinant protein.
Releasing Bacterial DNA
- Bacterial DNA can be released by lysing the cells using physical, chemical, or enzymatic methods to break the bacterial cell wall and membrane.
Uses of Cloning Vectors
- Cloning vectors are used to:
  - Propagate gene fragments.
  - Introduce foreign DNA into host cells.
  - Express proteins.
Agrobacterium tumefaciens as a Natural Genetic Engineer
- Agrobacterium tumefaciens is called a natural genetic engineer of plants because it transfers a portion of its DNA (T-DNA) into the plant genome, facilitating the incorporation of new genetic material into the plant cells.
Selectable Marker in Cloning Vectors
- A selectable marker is essential to identify and select cells that have successfully incorporated the recombinant DNA. Without it, distinguishing transformed from non-transformed cells is difficult.
Eukaryotic Cells and Restriction Endonucleases
- Eukaryotic cells do not typically have restriction endonucleases because these enzymes are generally produced by bacteria as a defense mechanism against foreign DNA.
cDNA Libraries
- cDNA libraries are collections of complementary DNA synthesized from mRNA. cDNA is made through reverse transcription and represents the expressed genes in a particular cell type.
Thermus aquaticus in Recombinant DNA Technology
- Thermus aquaticus is utilized due to its heat-stable DNA polymerase (Taq polymerase), which is essential for polymerase chain reaction (
  PCR) because it can withstand high temperatures during denaturation of DNA.
Molecular Scissors
- Molecular scissors refer to restriction enzymes, which cut DNA at specific sites. An example is EcoRI.
  - Role in Recombinant DNA Technology: They create cut sites for inserting foreign DNA into vectors.
Agrobacterium tumefaciens as a Cloning Vector
- Agrobacterium tumefaciens functions as a cloning vector due to its natural mechanism to transfer DNA to plant cells, making it a powerful tool for genetic engineering in plants.
Introduction of Recombinant DNA into Host Cells
- Recombinant DNA can be introduced into host cells through various methods (e.g., electroporation, heat shock), which transiently permeabilize the cell membrane to allow DNA entry.
Thermus aquaticus in Gene Amplification
- Thermus aquaticus contributes through its heat-stable polymerase that allows for consistent and efficient amplification of target genes during the PCR process.
Recombinant Proteins and Bioreactors
- Recombinant proteins are proteins produced from recombinant DNA technologies. Bioreactors provide controlled environments for efficiently producing these proteins at scale.
DNA Isolation from Bacterial Cells
- DNA isolation involves lysing the bacterial cells and applying techniques such as phenol-chloroform extraction, alcohol precipitation, or silica column purification.
Techniques for DNA Fragment Isolation
- Techniques include gel electrophoresis for separation and purification, and methods like PCR for amplification of specific DNA fragments.
Source of Taq Polymerase
- Taq polymerase is sourced from Thermus aquaticus. Its advantage includes thermal stability, allowing for repeated temperature cycling in PCR.
Ti Plasmid Source
- The Ti plasmid is isolated from Agrobacterium tumefaciens and is used in biotechnology for transferring genes into plant cells.
EcoRI
- EcoRI is a restriction enzyme that recognizes the specific palindromic sequence. The ‘R’ stands for the strain of E. coli from which it was isolated (RY13).
Recombinant DNA Formation with EcoRI
- To form recombinant DNA using EcoRI, the enzyme cuts both vector DNA and foreign DNA at specific palindromic sequences to generate sticky ends that enable joining the two DNAs together.
Formation of Sticky Ends in Recombinant DNA
- Sticky ends are created when restriction enzymes like EcoRI cut at specific sites, leaving overhanging single-stranded DNA ends that facilitate ligation with complementary sequences.
Primers in PCR
- (i) Two primers are required for each cycle of PCR. Primers anneal to specific sequences on the template DNA, enabling DNA polymerase to synthesize new strands.
- (ii) The DNA polymerase must be heat-stable, typically from Thermus aquaticus.
Action of EcoRI
- EcoRI recognizes the sequence GAATTC and cuts between the G and A nucleotides, forming sticky ends on both strands of the DNA.
Visualization of DNA Fragments in Gel Electrophoresis
- DNA fragments separated by gel electrophoresis can be visualized using UV light after staining with ethidium bromide, which intercalates between the bases of DNA and fluoresces under UV light.
Making Bacterial Cells Competent
- Bacterial cells can be made competent by treatment with calcium ions or by heat shock or electroporation methods to allow DNA uptake.
Origin of Replication (Ori)
- The origin of replication is essential for a cloning vector as it ensures that the vector is replicated within the host organism, producing multiple copies.
Key Tools in Recombinant DNA Technology
- Key tools include:
  - Restriction enzymes
  - DNA ligase
  - Vectors (e.g., plasmids)
  - Selectable markers
  - Polymerases
Role of Ti Plasmids
- Ti plasmids are used in plant biotechnology for transferring genes, facilitating plant transformation and integration of foreign DNA into the plant genome.
Cohen and Boyer's Work
- Cohen and Boyer developed techniques for creating recombinant DNA by joining DNA from two different organisms using restriction enzymes and ligase, which resulted in foundational technology for genetic engineering.
Role of DNA Ligase
- DNA ligase is crucial for joining DNA fragments together by forming phosphodiester bonds between adjacent nucleotides, allowing for the creation of stable recombinant DNA molecules.
Selection of Recombinant Colonies
- (a) Selection can be accomplished by introducing a gene insertion that disrupts a colony color marker, allowing for easy identification of recombinant colonies.
- (b) This process is known as “insertional inactivation” because the insertion of the foreign gene inactivates a specific gene in the vector, leading to observable phenotypic changes.
Source of Taq Polymerase
- Taq polymerase is sourced from Thermus aquaticus, which is notable for its ability to withstand high temperatures during PCR processes, maintaining high enzyme activity.
Steps for Biotechnology Experiments
- (a) Isolation of DNA from bacterial cells involves lysing cells and purifying DNA through chemical or physical means.
- (b) Reintroduction of recombinant DNA involves transformation methods such as heat shock or electroporation to allow uptake by competent bacterial cells.
Amplification of Gene Samples using PCR
- Gene amplification using PCR is carried out by repeated cycles of denaturation, annealing of primers, and extension by DNA polymerase to exponentially increase the quantity of the target DNA sequence.
Naming a Restriction Endonuclease
- The naming of a restriction endonuclease follows a combination of the genus description, species name, and strain designation. For example, EcoRI is named after Escherichia coli RY13.
Agrobacterium tumefaciens as Cloning Vector
- Agrobacterium tumefaciens has been modified to facilitate the transport of foreign DNA into plant cells by utilizing its natural transformation abilities, promoting genetic engineering in plants.

Long Answer Questions

Role of Vectors in Recombinant DNA Technology
- (a) Vectors are essential for the transport of genetic material into host organisms and maintenance of that genetic material within those cells. Examples are plasmids and viral vectors.
- (b) Diagrammatic representation includes:
  1. Isolation of DNA fragment.
  2. Cutting of plasmid vector.
  3. Ligation of DNA fragment into vector.
  4. Transformation of host cells with the recombinant vector.
Engineered Vectors for Gene Transfer
- (a) Engineered vectors are preferred by biotechnologists because they can be tailored for specific features enhancing efficiency in gene transfer.
- (b) “ori” provides the replication initiation point; “selectable markers” allow for identification of successfully transformed cells; “cloning sites” are where foreign DNA is inserted for manipulation.
Bioreactor Functionality
- A bioreactor is a controlled environment where biological reactions occur.
- A sparged stirred bioreactor includes mechanisms for aeration and mixing to optimize growth conditions for microorganisms or cells and promote product synthesis.
  - Diagram includes: broth, sparger, stirring mechanism, and gas inlet/outlet.
Characteristics of a Cloning Vector
- (i) A cloning vector must possess a selectable marker, origin of replication, and multiple cloning sites.
- (ii) DNA cannot pass through the cell membrane because it is hydrophilic. Bacterial cells can be made competent through calcium treatment or electroporation, facilitating DNA uptake.
Process of Cutting Desired Gene
- (i) The desired gene can be cut using restriction endonucleases at specific sites identified by palindromic sequences.
- (ii) Synthesis of multiple copies is accomplished through PCR, utilizing primers and DNA polymerase for amplification.
Formation of Recombinant DNA by EcoRI
- (a) Diagrammatically show that EcoRI cuts DNA at GAATTC sequences, creating sticky ends.
- (b) Gel electrophoresis is used for separating fragments of DNA cut by the restriction endonuclease.
Formation of Sticky Ends
- Sticky ends are created when restriction enzymes cut DNA at specific palindromic sequences resulting in overhangs. They are called sticky due to their ability to anneal with complementary DNA sequences easily.
Role of Ori and Restriction Site in pBR322
- The Ori facilitates replication of the plasmid within the host, while the restriction sites enable the insertion of foreign DNA fragments, essential for cloning processes in biotechnology.