Table of Contents (tap to open/close)
Principles of Biotechnology
This chapter Biotechnology: Principles and Processes, focuses on the fundamental principles and detailed processes involved in biotechnology, with a specific emphasis on recombinant DNA technology and its tools.
Introduction to Biotechnology
- Definition: Using live organisms or their enzymes to produce useful products.
- Definition by EFB (European Federation of Biotechnology) : Biotechnology is the combination of natural science with organisms or their parts to create products and services.
- Examples: Making curd, bread, wine.
- Modern Biotechnology: Involves genetically modified organisms for large-scale production.
- Examples: Test-tube babies (in vitro fertilization), gene synthesis, DNA vaccines, gene therapy.
Principles of Biotechnology
Two Core Techniques:
- Genetic Engineering
- Alters genetic material (DNA and RNA).
- Introduces modified DNA into host organisms.
- Changes the host organism’s characteristics.
- Bioprocess Engineering
- Maintains sterile environments for microbial (host) growth.
- Produces products like antibiotics, vaccines, and enzymes on a large scale.
Recombinant DNA Technology
History
Basic Steps & Concepts in Genetically Modifying an Organism
1. Identification of DNA with Desirable Genes
- Traditional Methods drawbacks:
- Sexual reproduction adds variation; asexual reproduction preserves genes and hybridization can add undesirable genes along with desired ones. So these are the limitations of traditional methods.
- Genetic Engineering benefits:
- Allows the isolation and introduction of only desirable genes, avoiding the limitations of traditional methods.
- So with genetic engineering, focus on identifying and isolating the specific gene you want to introduce. i.e. desirable gene.
- Note- Different synonyms have been used for the desirable gene throughout the chapter, such as Gene of interest, Target gene, Foreign (alien) gene, Source DNA/gene, and Inserted gene. All imply the same meaning.
2. Introduction of Identified DNA into the Host
- Vector DNA (e.g., plasmid) is used to deliver alien DNA into the host organism.
3. Maintenance of Introduced DNA in the Host and Transfer to Progeny
- Alien DNA lacks the origin of replication (ori) sequence needed for self-replication.
- Integration into the host genome, which contains the ori sequence, ensures that the alien DNA can replicate (cloning) and be inherited alongside the host DNA.
- cloning = making multiple identical copies of DNA.
Recombinant DNA Technology
- Involves joining and inserting foreign DNA into a host organism to produce new genetic combinations.
Detailed Sequential Process (steps): have a look!
Tools of Recombinant DNA Technology
To perform genetic engineering or recombinant DNA technology, we need key tools like
- Enzymes (restriction enzymes, polymerase enzymes, ligases)
- Vectors (DNA carrying vehicle)
- Host organisms(bacterial, plant, or animal cell).
Let’s understand some of these in detail.
1. Restriction Enzymes (R.E)/molecular scissors
- Definition: Restriction enzymes are nucleases that cut DNA at specific sites into fragments.
- Discovery: In 1963, two enzymes that restrict bacteriophage growth in Escherichia coli were discovered. One added methyl groups to DNA, while the other cut DNA (restriction endonuclease).
- First Restriction Enzyme: Hind II, identified in 1968, cuts DNA at specific sequences.
- Today, over 900 restriction enzymes are known.
- Naming: Restriction enzymes are named based on the bacteria they come from, e.g., EcoRI is from Escherichia coli.
- The first letter indicates the genus, and the next two letters indicate the species of the prokaryotic cell from which they were isolated.
- E.g. EcoRI comes from E. coli RY 13 (R = the strain. Roman numerals indicate the order in which the enzymes were isolated from that strain of bacteria).
- Types of Nucleases (R.E):
- Exonucleases: Remove nucleotides from DNA ends.
- Endonucleases: Cut DNA at specific internal positions.
- Function: Restriction endonucleases inspect DNA and cut at specific palindromic sequences (e.g., GAATTC), creating sticky ends.
- Palindromic sequences : Sequences that read the same backward and forward (e.g., 5′ —— GAATTC —— 3′ and 3′ —— CTTAAG —— 5′).
- This palindromic sequence is recognized only by EcoRI and is also called the recognition sequence for the enzyme.
- Sticky Ends:
- When restriction enzymes cut DNA, these cuts create single-stranded overhangs called sticky ends.
- These overhanging (sticky) ends can form hydrogen bonds with complementary sequences, for easy joining of complementary sequences.
- Sticky ends ensure fragments can be joined with DNA ligases if cut by the same enzyme.
- Final Result: Creates recombinant DNA by combining DNA from different sources.
Separation and Isolation of DNA Fragments
Gel Electrophoresis
- Purpose: Separates DNA fragments based on size.
- Process:
- DNA Charge and Movement: DNA fragments being negatively charged move towards the anode (+ve charge) in an electric field through agarose gel.
- Separation by Size/Sieving effect of the agarose gel: This above movement separate DNA fragments by size (smaller fragments move farther).
- Agarose gel, which acted as a matrix, extracted from seaweeds,
- Visualization:
- DNA is stained with ethidium bromide and viewed under UV light.
- Bright orange bands indicate DNA fragments.
- Elution: DNA bands are cut from the gel and extracted for further use.
Summary of Steps
- Cutting DNA: Restriction enzymes cut DNA at specific sites.
- Separating DNA: Gel electrophoresis separates DNA fragments by size.
- Visualizing DNA: Ethidium bromide staining and UV light are used to see DNA bands.
- Extracting DNA: Elution removes the DNA bands from the gel for use in recombinant DNA construction.
By using these tools, scientists can create recombinant DNA molecules, which are combinations of DNA from different sources, to study and use in various applications.
2. Cloning Vectors
- Definition: Cloning vectors are DNA molecules used to carry foreign DNA into a host cell.
- Example: Plasmids and bacteriophages are commonly used as vectors because they can replicate independently within bacterial cells.
- Plasmids are autonomously replicating, circular DNA found in bacteria, existing as extra-chromosomal DNA.
- Plasmids have 1-100 copies per cell; bacteriophages have even more.
- When cloning vectors are multiplied in the host, the attached piece of foreign DNA is also replicated.
- Thus linking foreign DNA to these vectors allows for multiplying (cloning) the foreign DNA.
Key Features of Cloning Vectors
- 1. Origin of Replication (ori):
- Definition: A specific DNA sequence where the replication process begins.
- Function: Any DNA (foreign) linked to this (ori) can replicate in the host cell.
- This ensures that the inserted DNA is duplicated along with the host’s own DNA.
- Control of Copy Number: Ori also determines how many copies of the linked DNA are made.
- Some origins of replication lead to the production of many copies, while others produce only a few.
- Importance: Ensures multiple copies of target DNA are available for further use.
- 2. Selectable Marker:
- Definition: A selectable marker is a gene that helps identify and distinguish transformants (cells that have taken up the vector) from non-transformants (cells that haven’t), enabling the elimination of non-transformants.
- Function: Allows growth of transformants by providing a selectable advantage, such as antibiotic resistance.
- Example:
- Antibiotic Resistance Genes: Common markers include genes for resistance to antibiotics like ampicillin, chloramphenicol, tetracycline, or kanamycin.
- Color-Producing Genes: Sometimes, genes for producing colors are also used as markers.
Explanation: Antibiotic Resistance Markers:
- 3. Cloning Sites:
- Definition: Cloning sites are basically the recognition sites for restriction enzymes. They are essential for linking foreign DNA to vectors.
- Single Site Preference: Preferably only one site is required to avoid complications from unwanted cuts.
- because multiple sites can lead to unwanted cuts in the vector, making the cloning process less efficient and increasing the risk of inserting the foreign DNA in the wrong location.
- Example: pBR322 plasmid has sites like BamH I (pronounced “Bam H one”) in the tetracycline resistance gene.
- Ligation of foreign DNA here disturbs this site and inactivates tetracycline resistance, making selection of recombinants easier.
Insertional Inactivation and Selection of Recombinant Plasmids
- Definition: When foreign DNA is inserted into a bacterial gene, it inactivates/disturb that gene—a process known as insertional inactivation.
- For instance in above example, recombinant plasmids lose tetracycline resistance when foreign DNA is inserted.
- Methods of Insertional Inactivation (2 ways):
- Deactivating an Antibiotic Resistance Gene
- Deactivating a Chromogenic (color producing) Gene
- A. Selection Method Using Antibiotic Resistance:
- Recombinant plasmids lose resistance to one antibiotic due to the insertion of foreign DNA.
- Selection is done by growing cells on media with different antibiotics.
- Transformants grow on ampicillin media but not on tetracycline media.
- Non-recombinants grow on both.
Detailed Example- Antibiotic Resistance Method
- B. Selection Method Using Chromogenic Substrates:
- This method differentiates recombinants using a chromogenic substrate.
- Mechanism: Insertion of foreign DNA into the β-galactosidase gene inactivates it.
- Selection:
- Recombinants: Having recombinant plasmids, do not produce color (white/no color colonies.
- Non-recombinants: Retain β-galactosidase activity (functional gene), producing blue colonies in the presence of a chromogenic substrate.
4. Vectors for Cloning Genes in Plants and Animals
- Learning from Nature:
- Bacteria and viruses naturally deliver their genes to eukaryotic cells in order to infect them.
- Example 1: Agrobacterium tumifaciens, a pathogen of dicot plants, uses T-DNA to turn plant cells into tumors for its benefit.
- Example 2: Retroviruses in animals can turn normal cells into cancerous cells.
- Conclusion: So, these can be used to deliver desirable genes into animal cells.
- Transforming Pathogens into Tools/Using Pathogens as Vectors:
- Scientists create modified/disarmed, non-virulent vectors from pathogens to deliver genes of interest into plants, utilizing their delivery mechanisms without causing disease.
- Ti Plasmid: Modified from Agrobacterium tumifaciens, no longer pathogenic, used to deliver genes into plants.
- Disarmed Retroviruses: Used to deliver genes into animal cells.
- Process:
- Gene/DNA fragment is ligated into a vector to make recombinant.
- This recombinant is transferred into a bacterial, plant, or animal host for multiplication.
3. Competent Host for Transformation with Recombinant DNA
- Why can’t DNA enter cells easily?
- DNA is hydrophilic and can’t pass through cell membranes easily or on its own.
- Bacterial cells need to be made “competent/capable” to take up DNA.
- Methods for making Bacteria cell Competent:
- Treat with Divalent Cation:
- Treat bacterial cells with a divalent cation like calcium.
- This helps DNA enter the bacterium through pores in the cell wall.
- Heat Shock Method:
- Incubate bacterial cells with recombinant DNA on ice.
- Briefly heat them at 42°C (heat shock).
- Return to ice for cooling.
- Bacteria take up recombinant DNA.
- Treat with Divalent Cation:
- Other Methods to Introduce DNA:
- Micro-injection: Directly inject recombinant DNA into the nucleus of an animal cell or bacterial cell.
- Biolistics/Gene Gun: High velocity micro-particles of gold or tungsten coated with DNA are bombarded into plant cells.
- Disarmed Pathogen Vectors: Use modified pathogens to transfer recombinant DNA into host cells. e.g. A. tumefaciens.
These tools and methods help scientists in genetic engineering by allowing the easy identification and multiplication of desired DNA sequences within different host cells.
Processes of Recombinant DNA Technology
Recombinant DNA technology involves several steps in a specific order:
- Isolation of DNA
- Fragmentation of DNA
- Isolation of Desired DNA Fragment
- Ligation of DNA Fragment into a Vector
- Transferring Recombinant DNA into the Host
- Culturing Host Cells on a Large Scale
- Extraction of the Desired Product
1. Isolation of the Genetic Material (DNA)
- Genetic Material: DNA is the genetic material in most organisms.
- Breaking Cells: To get pure DNA, cells (cell wall) must be broken open.
- Enzymes Used to break cell wall:
- Lysozyme for bacteria
- Cellulase for plant cells
- Chitinase for fungus
- Removing Other Molecules:
- Purpose- DNA must be pure and free from other macromolecules.
- RNA is removed by ribonuclease.
- Proteins are removed by protease.
- Purpose- DNA must be pure and free from other macromolecules.
- Purifying DNA: After removing other molecules, chilled ethanol is added to precipitate the DNA, which appears as fine threads.
2. Cutting of DNA at Specific Locations
- Restriction Enzymes: Used to cut DNA at specific sites.
- Agarose Gel Electrophoresis: Checks the progress of DNA cutting. DNA moves towards the positive electrode (anode) because it is negatively charged. (detailed process of gel electrophoresis given above).
- Preparing Recombinant DNA:
- Both source DNA and vector DNA are cut with a specific restriction enzyme.
- The ‘gene of interest’ from the source DNA is mixed with the cut vector DNA.
- Ligase is added to join the DNA fragments, creating recombinant DNA.
This process allows scientists to combine DNA from different sources and produce desired products.
3. Amplification of Gene of Interest using PCR
- PCR (Polymerase Chain Reaction): A technique to make many copies of a specific/desired DNA segment in lab (in vitro).
- Components:
- Primers: Small DNA pieces (oligonucleotides) that match the start and end (complementary) of the target DNA.
- DNA Polymerase: Enzyme that extends the primers to create new DNA strands.
- Process:
- Denaturation: Heating the target DNA at 94°C to separate the strands, which then act as templates for DNA synthesis.
- Annealing: Joining the two primers at 52°C to the 3′ ends of the DNA templates.
- Extension: Adding nucleotides to the primers using Taq polymerase, which remains active at high temperatures during denaturation.
- DNA (Taq) polymerase uses the primers and nucleotides to replicate the DNA.
- Repeating these processes amplifies the DNA segment up to a billion times and produce billion copies.
- Thermostable DNA Polymerase: A thermostable DNA polymerase i.e. Taq polymerase, from Thermus aquaticus is used for above mentioned polymerization as it remains active even at high temperatures (during denaturation).
- Usage: The amplified DNA can be joined with a vector for further cloning.
4. Insertion of Recombinant DNA into the Host Cell/Organism
Purpose: To introduce ligated DNA into a recipient (host) cell or organism.
- Introduction Methods: Various methods (as discussed above) are used to introduce ligated DNA into recipient cells.
- Making Cells Competent: Recipient cells are treated to make them capable of taking up DNA.
- Example:
- A recombinant DNA with an antibiotic resistance gene (e.g., ampicillin resistance) is introduced into E. coli cells.
- Transformed cells gain resistance to ampicillin.
- When these cells are spread on agar plates with ampicillin, only transformed cells grow, while others die.
- Selectable Marker:
- Definition: A gene providing resistance to an antibiotic, like ampicillin.
- Function: Selectable markers enable the selection of transformed cells in the presence of specific antibiotics.
- Example: The ampicillin resistance gene helps in identifying transformed cells.
5. Obtaining the Foreign Gene Product
Purpose: To produce a desirable protein using recombinant DNA technology.
Process:
- Cloning and Expressing Genes:
- Insert foreign DNA into a cloning vector and transfer it into a host cell (bacterial, plant, or animal).
- Foreign DNA multiplies and expresses the desired (recombinant) protein.
- Recombinant Protein:
- Definition: A protein produced from a gene expressed in a different host.
- Culture and Extraction: Cells with foreign genes are grown in the lab, and the desired protein is extracted and purified.
Large-Scale Production:
- Continuous Culture System:
- Used for sufficient quantities of the desired protein.
- Small-scale cultures used in the lab for extraction and purification.
- Continuous systems replace old medium with fresh medium, maintaining optimal growth and higher protein yields.
Bioreactors:
- Purpose: Vessels/Tanks used for large-scale production (100-1000 liters).
- Function: Convert raw materials into specific products using cells.
- Provide optimal growth conditions (temperature, pH, oxygen).
- Example: Stirred-tank bioreactors.
- They are cylindrical or have a curved base for even mixing and oxygen availability.
- Components:
- Agitator system for mixing
- Oxygen delivery system
- Foam control system
- Temperature control system
- pH control system
- Sampling ports for withdrawing culture samples
6. Downstream Processing
Downstream Processing = Post-Biosynthesis Steps
Purpose: After producing the desired product, product needs further processing and undergoes separation and purification (downstream processing).
Steps Involved:
- Separation and Purification:
- Involves a series of steps to isolate the desired product
- These steps make sure the product is clean and pure.
- Preparation for Market:
- Formulation: The product is mixed with preservatives to keep it stable.
- Clinical Trials: For drugs, the product must be tested thoroughly to ensure it is safe and effective.
- Quality Control: Strict checks ensure the product meets standards.
Summary
- Downstream processing ensures the product is safe, pure, and ready for use.
- It includes steps like purification, adding preservatives, clinical trials, and quality checks.
Chapter Summary
- Biotechnology involves making and selling products using live organisms, cells, or enzymes.
- Modern biotechnology uses genetically modified organisms.
- This became possible when we learned to change DNA and make recombinant DNA.
- This key process is called recombinant DNA technology or genetic engineering.
- It involves:
- Using restriction endonucleases and DNA ligase.
- Using plasmid or viral vectors to carry foreign DNA into host organisms.
- Expressing the foreign gene.
- Purifying the gene product (functional protein).
- Formulating the product for marketing.
- Large-scale production uses bioreactors