Biotechnology: Principles and Processes
Biotechnology is the application of biological techniques to solve problems and create products for human benefit.
Principles of Biotechnology
- Recombinant DNA Technology: Joining DNA fragments from different sources to create new DNA molecules.
- Cloning: Producing identical copies of an organism or DNA sequence.
Tools of Biotechnology
- Cloning Vectors: Plasmids or viruses used to carry foreign DNA into a host cell.
- Gel Electrophoresis: Separates DNA fragments based on size.
- Polymerase Chain Reaction (PCR): Amplifies DNA fragments.
Applications of Biotechnology
- Agriculture: Developing genetically modified crops with improved traits, such as pest resistance and higher yields.
- Medicine: Producing vaccines, antibiotics, and therapeutic proteins.
- Environmental Biotechnology: Bioremediation of pollution and waste management.
- Industrial Biotechnology: Production of enzymes, chemicals, and biofuels.
Key Techniques:
- Genetic Engineering: Altering the genetic makeup of organisms.
- Hybridization: Crossing different species to create hybrids with desirable traits.
- Tissue Culture: Growing cells or tissues in a controlled environment.
Exercise :
1. Can you list 10 recombinant proteins which are used in medical practice? Find out where they are used as therapeutics (use the internet).
Ans :
| Recombinant Protein | Therapeutic Use |
| Insulin | Treatment of type 1 diabetes mellitus |
| Growth hormone | Treatment of growth hormone deficiency |
| Erythropoietin (EPO) | Treatment of anemia, especially in patients with kidney disease |
| Factor VIII | Treatment of hemophilia A |
| Factor IX | Treatment of hemophilia B |
| Interferon-alpha | Treatment of chronic hepatitis B and C, multiple sclerosis, and certain types of cancer |
| Interferon-beta | Treatment of multiple sclerosis |
| G-CSF (Granulocyte colony-stimulating factor) | Treatment of neutropenia (low white blood cell count), often after chemotherapy or bone marrow transplantation |
| GM-CSF (Granulocyte-macrophage colony-stimulating factor) | Treatment of severe infections and bone marrow transplant complications |
| Tissue plasminogen activator (tPA) | Treatment of acute myocardial infarction (heart attack) and stroke |
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.
Ans :
3. From what you have learnt, can you tell whether enzymes are bigger or DNA is bigger in molecular size? How did you know?
Ans :
Enzymes are typically larger than DNA molecules.
Here’s how we can determine this:
- Molecular Weight: Enzymes are proteins, which are composed of multiple amino acids linked together by peptide bonds. Proteins generally have higher molecular weights compared to DNA, which is composed of nucleotides.
- Structure: Enzymes have complex three-dimensional structures with multiple domains and active sites, while DNA has a relatively simpler double-helical structure.
- Function: They are often larger and more complex than the DNA molecules they interact with.
4. What would be the molar concentration of human DNA in a human cell? Consult your teacher
Ans : The molar concentration of DNA in human cell is 2 mg/ml of cell extract.
5. Do eukaryotic cells have restriction endonucleases? Justify your answer.
Ans :
No
They are primarily found in bacteria, where they serve as a defense mechanism against invading viruses. These enzymes cleave the viral DNA, preventing the virus from replicating.
Eukaryotic cells do not need restriction endonucleases for the same purpose because they have their own immune systems to combat viral infections. Instead, eukaryotic cells rely on other mechanisms, such as the production of antibodies and the activation of immune cells, to defend against viruses.
6. Besides better aeration and mixing properties, what other advantages do stirred tank bioreactors have over shake flasks?
Ans :
Here are some additional advantages of stirred tank bioreactors over shake flasks:
- Larger Scale Production: Stirred tank bioreactors can handle much larger volumes of culture than shake flasks, making them suitable for industrial-scale production of biomolecules.
- Improved Control: Stirred tank bioreactors allow for precise control of various parameters such as temperature, pH, dissolved oxygen, and nutrient levels, which is critical for optimal growth and product yield.
- Uniformity: The stirring mechanism ensures better mixing and distribution of nutrients and oxygen throughout the culture, leading to more uniform growth conditions.
- Scalability: Stirred tank bioreactors can be easily scaled up to meet increasing production demands, while shake flasks are limited in size.
- Automation: Many stirred tank bioreactors can be automated, reducing the need for manual labor and ensuring consistency in the production process.
- Sterility: Stirred tank bioreactors can be designed to maintain a sterile environment, which is essential for preventing contamination and ensuring the quality of the product.
7. Collect 5 examples of palindromic DNA sequences by consulting your teacher. Better try to create a palindromic sequence by following base-pair rules.
Ans :
ere are 5 examples of palindromic DNA sequences:
- GATATC
- ACGTACGT
- MADAM (when written in DNA base codes: ATAT)
- RACECAR (when written in DNA base codes: AGTCGT)
- ROTATOR (when written in DNA base codes: ATGCGTA)
Creating a Palindromic Sequence:
To create a palindromic sequence, simply write a sequence of nucleotides in the 5′ to 3′ direction, and then reverse it to get the complementary strand. For example:
- Original sequence: 5′-AATCG-3′
- Complementary strand: 3′-TTAGC-5′
- Palindromic sequence: 5′-AATCGGAATTC-3′
8. Can you recall meiosis and indicate at what stage a recombinant DNA is made?
Ans :
Meiosis is a type of cell division that produces gametes (sperm or egg cells) with half the number of chromosomes as the original cell.
Recombinant DNA is formed during meiosis I, specifically in the crossing-over stage. This process occurs during the first meiotic division, when homologous chromosomes pair up and form tetrads.
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?
Ans :
Reporter enzymes can be used to monitor the transformation of host cells by foreign DNA in addition to a selectable marker by providing a visual or biochemical signal that indicates successful transformation
10. Describe briefly the following:
(a) Origin of replication (b) Bioreactors (c) Downstream processing
Ans :
(a) Origin of replication: This is a specific DNA sequence on a plasmid or chromosomal DNA where DNA replication initiates. It serves as a binding site for replication proteins, allowing the DNA polymerase enzyme to start copying the DNA strand.
(b) Bioreactors: These are vessels or containers used for carrying out biological processes on a large scale. They provide a controlled environment for cultivating microorganisms or cells, and can be used for various applications such as producing pharmaceuticals, enzymes, and biofuels.
(c) Downstream processing: This refers to the steps involved in isolating, purifying, and formulating a product produced by a biological process. It typically involves a series of operations like filtration, centrifugation, chromatography, and drying.
11. Explain briefly
(a) PCR (b) Restriction enzymes and DNA (c) Chitinase
Ans :
(a) PCR (Polymerase Chain Reaction): A technique used to amplify DNA fragments exponentially.
(b) Restriction Enzymes and DNA: Restriction enzymes are enzymes that cut DNA at specific recognition sequences known as restriction sites. They are used in molecular biology to manipulate DNA fragments and create recombinant DNA.
(c) Chitinase: An enzyme that breaks down chitin, a polysaccharide found in the cell walls of fungi and insects. Chitinase is used in various applications, such as biofuel production and the treatment of fungal infections.
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
Ans :
(a) Plasmid DNA and Chromosomal DNA
| Feature | Plasmid DNA | Chromosomal DNA |
| Location | Extrachromosomal (outside the chromosome) | Within the chromosome |
| Size | Smaller than chromosomal DNA | Larger than plasmid DNA |
| Replication | Independent of chromosomal replication | Replicates along with chromosomal DNA |
| Copy Number | Can exist in multiple copies within a cell | Typically present in a single copy |
| Function | Often carries genes for antibiotic resistance, virulence factors, or other traits | Contains essential genes for the organism’s survival |
(b) RNA and DNA
| Feature | RNA | DNA |
| Sugar | Ribose | Deoxyribose |
| Nitrogenous Bases | Adenine, guanine, cytosine, uracil | Adenine, guanine, cytosine, thymine |
| Structure | Single-stranded | Double-stranded |
| Function | Messenger (mRNA), transfer (tRNA), ribosomal (rRNA); also involved in gene regulation | Stores genetic information and serves as a template for DNA replication and transcription |
(c) Exonuclease and Endonuclease
| Feature | Exonuclease | Endonuclease |
| Cleavage Site | Cleaves nucleotides from the ends of a DNA strand | Cleaves nucleotides within a DNA strand |
| Function | Involved in DNA repair and replication | Used in molecular biology techniques, such as restriction enzyme digestion and DNA sequencing |
| Examples | DNA polymerase I (3’→5′ exonuclease activity) | EcoRI, HindIII |
