Biotechnology: Principles and Processes Study Notes

Context and History of Biotechnology

Anthropocentric Approach: Since the era of Rene Descartes (a seventeenth-century French philosopher, mathematician, and biologist), human knowledge and natural sciences have been focused on developing technologies to enhance creature comforts and add value to human life.
Physics and Chemistry: These disciplines gave rise to engineering and industries dedicated to human welfare.
Biology: Historically, the primary utility of the biological world was as a food source. Modern biology shifted this in the twentieth century with the off-shoot of biotechnology.
Impact of Biotechnology: It has brought qualitative improvements in health and food production.

Herbert Boyer (1936 – Present)

Early Life: Born in 1936 in western Pennsylvania, an area where railroads and mines were the typical career paths for young men.
Academic Progression: * Completed graduate work at the University of Pittsburgh in 1963. * Conducted three years of post-graduate studies at Yale. * Assumed an assistant professorship at the University of California, San Francisco in 1966.
Key Discoveries (1969): Boyer studied restriction enzymes of the $E. coli$ bacterium. * Capability: He observed these enzymes could cut DNA strands in a specific fashion. * Sticky Ends: These cuts left overhanging portions known as ‘sticky ends.’ * Pasting DNA: These ends allowed for the precise exercise of pasting different pieces of DNA together.
Collaboration with Stanley Cohen: Cohen, a Stanford scientist, was studying plasmids (small ringlets of DNA that float freely in bacterial cytoplasm and replicate independently). * Plasmid Manipulation: Cohen developed methods to remove and reinsert plasmids into cells. * The Breakthrough: By combining DNA splicing (Boyer) with plasmid manipulation (Cohen), they could recombine DNA segments in desired configurations and insert them into bacteria. This turned bacteria into "manufacturing plants" for specific proteins.

Definition and Scope of Biotechnology

General Definition: Biotechnology involves techniques using live organisms or their enzymes to produce products and processes useful to humans.
Traditional Examples: Microbe-mediated processes like making curd, bread, or wine.
Modern Restricted Sense: Refers specifically to processes using Genetically Modified Organisms (GMOs) to achieve large-scale production.
Expanded Scope: Includes in vitro fertilisation (producing ‘test-tube’ babies), gene synthesis, DNA vaccine development, and correcting defective genes.
European Federation of Biotechnology (EFB) Definition: “The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.”

Principles of Modern Biotechnology

Genetic Engineering: Techniques used to alter the chemistry of genetic material ( $DNA$ and $RNA$ ) to introduce them into host organisms, thereby changing the host's phenotype.
Bioprocess Engineering: The maintenance of sterile (microbial contamination-free) environments in chemical engineering processes. This allows the growth of only the desired microbe or eukaryotic cell in large quantities for manufacturing products like antibiotics, vaccines, and enzymes.
Conceptual Development: * Sexual vs. Asexual Reproduction: Asexual reproduction preserves genetic information, while sexual reproduction permits variation, which can be beneficial to organisms and populations. * Traditional Hybridization: Often leads to the inclusion of undesirable genes alongside desired ones. * Genetic Engineering Advantages: Allows for the isolation and introduction of only one or a set of desirable genes without introducing undesirable ones.

The Mechanics of DNA Cloning

Fate of Alien DNA: A piece of DNA transferred into an alien organism usually cannot multiply unless integrated into the host's genome.
Origin of Replication (ori): A specific DNA sequence in a chromosome responsible for initiating replication. To multiply, alien DNA must be linked to an ‘ori’ sequence.
Definition of Cloning: The process of making multiple identical copies of any template DNA.
First Artificial Recombinant DNA (1972): Stanley Cohen and Herbert Boyer linked a gene encoding antibiotic resistance with a native plasmid of $Salmonella \, typhimurium$ . * Molecular Scissors: Restriction enzymes used to cut DNA at specific locations. * DNA Ligase: The enzyme that acts on cut DNA molecules to join their ends, creating a circular autonomously replicating DNA in vitro (recombinant DNA). * Host Organism: The recombinant DNA was transferred into $Escherichia \, coli$ , where it replicated using the host's $DNA \, polymerase$ .
Three Basic Steps in GM Organisms: 1. Identification of DNA with desirable genes. 2. Introduction of the identified DNA into the host. 3. Maintenance of introduced DNA and transfer to progeny.

Tools of Recombinant DNA Technology

Key Tools: Restriction enzymes, polymerase enzymes, ligases, vectors, and the host organism.
Restriction Enzymes: * Discovery: Isolated in 1963 from $E. coli$ ; one enzyme added methyl groups to DNA, the other (restriction endonuclease) cut DNA. * Hind II: The first restriction endonuclease discovered (isolated 5 years after 1963). It recognizes a specific sequence of six base pairs. * Recognition Sequence: The specific base sequence recognized by a restriction enzyme. * Current Statistics: Over $900$ restriction enzymes isolated from over $230$ bacterial strains. * Naming Convention: * 1st letter: Genus ( $E$ for $Escherichia$ ). * 2nd/3rd letters: Species ( $co$ for $coli$ ). * 4th letter: Strain ( $R$ for $RY \, 13$ ). * Roman Number: Order of isolation from that strain ( $I$ ). * Nucleases: * Exonucleases: Remove nucleotides from the ends of DNA. * Endonucleases: Make cuts at specific positions within the DNA. * Palindromes in DNA: Sequences of base pairs that read the same on the two strands when the orientation ( $5' \rightarrow 3'$ ) is kept the same. * Example: * $5' \text{--- GAATTC --- } 3'$ * $3' \text{--- CTTAAG --- } 5'$
Action of Restriction Endonucleases: They cut the sugar-phosphate backbones a little away from the center of the palindrome, leaving single-stranded "sticky ends" that facilitate ligation via hydrogen bonding.

Separation and Isolation of DNA Fragments

Gel Electrophoresis: Used to separate DNA fragments.
Charge: DNA is negatively charged and moves toward the anode (positive electrode).
Matrix: Agarose (natural polymer from seaweeds).
Sieving Effect: Fragments separate by size; smaller fragments move farther.
Visualization: DNA is stained with ethidium bromide and exposed to UV radiation, appearing as bright orange bands.
Elution: The process of cutting out the separated DNA bands from the agarose gel and extracting them.

Cloning Vectors

Plasmids and Bacteriophages: Used as vectors because they replicate independently of chromosomal DNA.
Copy Number: Bacteriophages have high copy numbers; plasmids range from $1-2$ to $15-100$ (or higher) per cell.
Required Features for Vectors: 1. Origin of Replication (ori): Controls the copy number of the linked DNA. 2. Selectable Marker: Helps identify and eliminate non-transformants. Examples: Genes for resistance to ampicillin, chloramphenicol, tetracycline, or kanamycin. Normal $E. coli$ lacks these resistances. 3. Cloning Sites: Preferably a single recognition site for commonly used restriction enzymes. Multiple sites complicate cloning. * Example: $pBR322$ allows ligation at the $BamH \, I$ site in the tetracycline resistance gene ( $tet^R$ ). Insertional inactivation causes the plasmid to lose tetracycline resistance while retaining ampicillin resistance ( $amp^R$ ). 4. Alternative Selectable Markers: Chromogenic substrates produce color. * Insertional Inactivation: Inserting DNA into the $\beta \text{-galactosidase}$ gene prevents enzyme production. Non-recombinants turn blue; recombinants remain colorless (white).
Vectors for Plants/Animals: * Agrobacterium tumifaciens: Uses the Ti-plasmid to deliver ‘T-DNA’ to plants, causing tumors. Modified into non-pathogenic cloning vectors. * Retroviruses: Disarmed and used to deliver genes into animal cells.

Competent Host and DNA Introduction

DNA Hydrophilicity: DNA cannot pass through cell membranes naturally.
Chemical Method: Treating bacteria with a divalent cation (e.g., $Ca^{2+}$ ) and applying heat shock ( $42^\circ \text{C}$ ) allows DNA to enter through wall pores.
Micro-injection: Recombinant DNA is injected directly into the nucleus of an animal cell.
Biolistics / Gene Gun: Cells (primarily plants) are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA.
Disarmed Pathogens: Vectors that transfer DNA upon infection.

Processes of Recombinant DNA Technology

Isolation of Genetic Material: * Cells broken using enzymes: Lysozyme (bacteria), Cellulase (plants), Chitinase (fungi). * Removal of contaminants: Ribonuclease for $RNA$ , Protease for proteins. * Precipitation: Purified $DNA$ precipitates as fine threads after adding chilled ethanol.
Cutting of DNA: Digestion with restriction enzymes at optimal conditions, checked by gel electrophoresis.
Amplification (PCR): Polymerase Chain Reaction. * Components: Primers (small oligonucleotides) and $DNA \, polymerase$ . * Mechanism: Denaturation, Annealing, and Extension. * Enzyme: $Taq \, polymerase$ from $Thermus \, aquaticus$ (thermostable). * Yield: Can produce $1 \, ext{billion}$ copies through $30$ cycles.
Ligation: Joining source DNA and vector DNA using ligase.
Insertion: Introducing DNA into a competent recipient (e.g., $E. coli$ ).
Obtaining Product: * Recombinant Protein: A protein-encoding gene expressed in a heterologous host. * Culture Systems: Small scale (lab) or continuous culture systems (fresh medium added, used medium drained to maintain log/exponential phase).
Bioreactors: Large vessels ( $100-1000 \, ext{liters}$ ) for processing raw materials into products. * Stirred-tank Reactor: Cylindrical with a curved base for mixing and oxygen availability. Features include agitator, oxygen delivery, foam control, temperature/pH control, and sampling ports.
Downstream Processing: Post-biosynthetic steps including separation, purification, formulation with preservatives, clinical trials (for drugs), and strict quality control.

Questions & Discussion

10 Recombinant Proteins in Medicine: Discuss therapeutics use (e.g., Insulin, Growth Hormone).
Enzyme vs. DNA Size: DNA molecules (long polymers) are generally much larger in molecular size than enzymes (proteins).
Eukaryotic Restriction Enzymes: Discussion on whether eukaryotes possess these (traditionally isolated from bacteria to restrict bacteriophages).
Stirred Tank vs. Shake Flask: Stirred tanks provide better aeration, mixing, and support larger volumes ( $100-1000 \, ext{liters}$ ).
Meiosis and Recombination: Identify the specific stage (Pachytene of Prophase I) where natural recombinant DNA is formed through crossing over.
Reporter Enzymes: Used to monitor transformation (similar to selectable markers) by providing a detectable signal (e.g., fluorescence or color change) upon gene expression.