Unit 5: Recombinant DNA Technology

Chapter 1: Principles of Genetic Engineering

(Excluding: hybridization techniques, DNA library, site-directed
mutagenesis)
1.1. Genetic Engineering Overview:
 Definition: Genetic engineering involves altering the genetic
makeup of an organism by introducing, eliminating, or rearranging
specific genes.
 Applications: Used in agriculture (e.g., genetically modified crops),
medicine (e.g., gene therapy, production of insulin), and industry
(e.g., production of enzymes).
 Objective: To give an organism desirable traits, like resistance to
pests in plants or the ability to produce human proteins.
1.2. Tools of Genetic Engineering:
 Restriction Enzymes: Enzymes that recognize specific sequences in
DNA and make precise cuts. This creates "sticky ends" or "blunt
ends" which allow for the insertion of foreign DNA. For example:
o EcoRI: Recognizes and cuts the sequence GAATTC.
o HindIII: Cuts at the sequence AAGCTT.
 DNA Ligase: After a DNA fragment is inserted into a vector, DNA
ligase joins the ends together, creating stable recombinant DNA.
 Vectors: Vehicles for introducing foreign DNA into host cells.
o Plasmids: Extra-chromosomal DNA found in bacteria, commonly
used in cloning.
o Bacteriophages: Viruses that infect bacteria, used as vectors for
large DNA fragments.
o Cosmids: Hybrid vectors that combine features of plasmids and
bacteriophages to carry larger DNA segments.
1.3. Recombinant DNA Technology:
 Process:
1. Isolation of Gene: The desired gene is isolated from the
donor organism.
2. Insertion into a Vector: The gene is inserted into a plasmid
or viral vector using restriction enzymes and DNA ligase.
3. Introduction into Host Cells: The recombinant DNA is
introduced into the host cell by transformation (in bacteria) or
transfection (in eukaryotic cells).
4. Selection of Transformed Cells: Selectable markers, such as
antibiotic resistance genes, help identify cells that have successfully
incorporated the recombinant DNA.
5. Expression of Gene: The host organism is grown under
conditions that promote the expression of the introduced gene,
producing the desired protein.
1.4. Transformation and Transfection:
 Transformation: A process where bacterial cells take up foreign
DNA from their surroundings. This is facilitated by making the
bacteria competent, usually by treating them with calcium chloride
or through electroporation.
o Competence: Refers to the ability of cells to take up foreign DNA.
Chemical methods make the cell membrane permeable, allowing
the uptake of plasmid DNA.
 Transfection: The introduction of foreign DNA into eukaryotic cells
(e.g., mammalian cells). Methods include:
o Chemical Methods: Use of calcium phosphate or liposomes.
o Physical Methods: Electroporation (electric pulse) or
microinjection.
o Viral Vectors: Using viruses to deliver the foreign DNA into the host
cells.
1.5. Selectable Markers:
 Antibiotic Resistance Genes: Common selectable markers include
genes for ampicillin resistance (amp^r) or kanamycin resistance.
These help in identifying successfully transformed cells.
 Reporter Genes: Genes like lacZ (which encodes β-galactosidase)
can be used to visually identify transformed cells through color
changes when grown on certain substrates.
1.6. Cloning and Expression of Recombinant DNA:
 Cloning: Once recombinant DNA is introduced into a host cell, the
host replicates the DNA, creating multiple copies of the inserted
gene. This process can be used for:
o Gene Cloning: Creating many copies of a particular gene.
o Protein Production: Producing large amounts of a specific protein
encoded by the cloned gene.
 Expression Vectors: Special types of vectors that contain the
necessary regulatory sequences (like promoters and terminators) to
ensure that the inserted gene is expressed in the host cell.
o Inducible Promoters: Can be activated under specific conditions,
allowing controlled expression of the inserted gene.
o Constitutive Promoters: Promote continuous expression of the
gene at all times.

Chapter 2: Manipulation of Nucleic Acids

(Excluding: 3-D shape of proteins, purification of proteins)
2.1. Extraction of Nucleic Acids:
 DNA Extraction:
o Cell Lysis: Cells are lysed using detergents or enzymes to break
down the cell membrane and release DNA.
o Removal of Proteins: Proteins are degraded by proteases or
separated by phenol-chloroform extraction.
o DNA Precipitation: DNA is precipitated using cold ethanol or
isopropanol and then spooled out or centrifuged to collect it.
 RNA Extraction:
o Use of RNase-free conditions: RNA is highly susceptible to
degradation, so extraction must be done in RNase-free
environments.
o Trizol Reagent: A common reagent for RNA extraction that
simultaneously lyses cells and inactivates RNases.
2.2. Gel Electrophoresis:
 Principle: DNA and RNA are negatively charged and move towards
the positive electrode when an electric current is applied. The
speed of migration depends on the size of the DNA or RNA
fragments, with smaller fragments moving faster.
 Agarose Gel: Used for the separation of larger DNA fragments,
typically in the range of 100 bp to 50 kb.
 Polyacrylamide Gel: Used for high-resolution separation of small
DNA or RNA fragments, typically below 1000 bp.
 Visualization: After electrophoresis, the gel is stained with ethidium
bromide or a fluorescent dye to visualize DNA under UV light.
2.3. Polymerase Chain Reaction (PCR):
 Components:
o Template DNA: The DNA that contains the target sequence.
o Primers: Short synthetic DNA sequences that flank the target
region.
o DNA Polymerase: A heat-stable enzyme, usually Taq polymerase,
used to extend primers and replicate the target DNA.
o Nucleotides (dNTPs): The building blocks of DNA.
o Thermal Cycler: A machine that controls the temperatures required
for the PCR cycles.
 Steps:
1. Denaturation (94-98°C): The double-stranded DNA is heated
to separate into single strands.
2. Annealing (50-65°C): The temperature is lowered to allow
the primers to bind (anneal) to their complementary sequences.
3. Extension (72°C): DNA polymerase extends the primers,
synthesizing new DNA strands complementary to the template.
o This cycle is repeated 25-35 times to amplify the target DNA
exponentially.
 Applications: PCR is used in cloning, gene expression analysis,
mutation detection, and forensic DNA analysis.
2.4. Blotting Techniques:
 Southern Blotting:
o Purpose: Detection of specific DNA sequences in a complex
mixture.
o Procedure: DNA is digested by restriction enzymes, separated by gel
electrophoresis, transferred to a membrane, and hybridized with a
labeled DNA probe.
 Northern Blotting:
o Purpose: Detection of specific RNA sequences.
o Procedure: Similar to Southern blotting, but RNA is separated by gel
electrophoresis.
 Western Blotting:
o Purpose: Detection of specific proteins.
o Procedure: Proteins are separated by SDS-PAGE, transferred to a
membrane, and detected using specific antibodies.

Chapter 3: Genomics and Bioinformatics

(Excluding: Introduction, genome sequencing projects, history of
bioinformatics, sequences and nomenclature)
3.1. Genomics Overview:
 Definition: Genomics is the study of the complete set of genes
(genome) in an organism, including their sequencing, mapping, and
functional analysis.
 Applications:
o Personalized Medicine: Tailoring treatments based on an
individual’s genetic profile.
o Agriculture: Developing genetically modified crops with improved
traits, such as resistance to pests or drought.
o Evolutionary Biology: Studying genetic variations and evolutionary
relationships between species.
3.2. Functional Genomics:
 Objective: To understand the function of genes and their products,
often by analyzing patterns of gene expression under different
conditions.
 Techniques:
o Gene Knockouts: Creating organisms in which specific genes have
been deleted or inactivated, helping to reveal the function of those
genes.
o Transcriptomics: Study of RNA transcripts to analyze gene
expression at a genome-wide level.
o Proteomics: The large-scale study of proteins, including their
structure, function, and interactions.
3.3. Comparative Genomics (continued):
 Objective: To compare the genomes of different species to
understand evolutionary relationships, gene conservation, and
functional importance.
o Conserved Genes: Genes that are preserved across different species
due to their essential roles in biological functions, such as genes
involved in DNA replication or cell division.
o Evolutionary Relationships: Comparative genomics helps identify
genetic similarities and differences between species, aiding in the
construction of phylogenetic trees, which represent evolutionary
links.
 Applications:
o Identifying Disease Genes: By comparing the human genome with
other species, researchers can identify genes associated with
hereditary diseases.
o Functional Annotation: Genomic comparisons can reveal the
functions of unknown genes by comparing them with similar
sequences in other organisms where the gene function is already
known.
3.4. Proteomics:
 Definition: The large-scale study of all proteins (proteome)
expressed by a genome, tissue, or cell.
 Importance: Proteins are the workhorses of the cell, carrying out
most of the biological processes, such as catalysis, transport, and
signaling.
 Techniques:
o Two-dimensional Gel Electrophoresis (2-DE): Proteins are first
separated based on their isoelectric point and then by molecular
weight, allowing for the analysis of complex protein mixtures.
o Mass Spectrometry (MS): Used to identify and quantify proteins by
measuring the mass-to-charge ratio of ionized protein fragments.
o Protein-Protein Interactions: Studied using techniques such as
yeast two-hybrid screening and co-immunoprecipitation, helping to
map interaction networks.
 Applications:
o Drug Development: Proteomics helps in identifying protein targets
for new drugs, understanding disease pathways, and monitoring
treatment responses.
o Biomarker Discovery: Identifying proteins that can serve as
indicators of disease, aiding in early diagnosis and monitoring.
3.5. Bioinformatics Tools:
 Definition: Bioinformatics involves the use of computational tools
to analyze, manage, and interpret biological data, particularly large
datasets like genomic sequences.
 Common Tools:
o BLAST (Basic Local Alignment Search Tool): Allows the comparison
of a query DNA or protein sequence against a database of
sequences to find similarities, aiding in gene function prediction and
evolutionary analysis.
o CLUSTAL: A program used for multiple sequence alignments,
important for studying conserved sequences across species.
o Genome Browsers: Software such as the UCSC Genome Browser or
Ensembl enables researchers to visualize genomic data, including
gene locations, regulatory elements, and variant data.
 Applications:
o Genetic Research: Analyzing large-scale genomic, transcriptomic,
and proteomic datasets to identify genes associated with diseases.
o Evolutionary Biology: Studying genetic divergence and conservation
across species.
o Structural Biology: Predicting the 3D structure of proteins based on
sequence data, aiding in drug design and understanding protein
function.

Unit 6: Microbial Biotechnology

Chapter 1: Microbial Production of Industrial Products
(Excluding: scale-up of microbial process)
1.1. Microbes in Industrial Biotechnology:
 Overview: Microorganisms such as bacteria, fungi, and yeast play a
crucial role in the production of various industrial products,
including enzymes, antibiotics, vitamins, organic acids, biofuels, and
biopolymers.
 Advantages of Using Microbes:
o Rapid Growth: Microbes reproduce quickly, allowing for large-scale
production in a short time.
o Genetic Manipulation: Microbes can be genetically engineered to
improve yields, enhance product quality, or produce new
substances.
1.2. Fermentation Processes:
 Definition: Fermentation is a metabolic process where microbes
convert sugars into organic acids, gases, or alcohol in the absence of
oxygen.
 Types of Fermentation:
o Alcoholic Fermentation: Carried out by yeast (e.g., Saccharomyces
cerevisiae), converting glucose to ethanol and carbon dioxide. Used
in brewing, wine-making, and biofuel production.
o Lactic Acid Fermentation: Performed by bacteria such as
Lactobacillus species, converting sugars into lactic acid, used in the
dairy industry for yogurt and cheese production.
o Mixed-Acid Fermentation: Microbes such as Escherichia coli
produce a mixture of organic acids (e.g., acetic acid, formic acid),
used in organic acid production.
 Fermentation Techniques:
o Batch Fermentation: Involves the growth of microorganisms in a
closed system where nutrients are supplied at the start, and the
process is stopped after product formation.
o Continuous Fermentation: Nutrients are continuously supplied, and
products are constantly removed, allowing for a steady-state
production.
o Fed-Batch Fermentation: A hybrid method where additional
nutrients are added over time without removing the culture, used
to maximize product yield.
1.3. Enzyme Production:
 Overview: Microbes are utilized for the production of industrial
enzymes, which catalyze chemical reactions in various sectors,
including food, pharmaceuticals, and biofuels.
 Examples:
o Amylase: Breaks down starch into simple sugars, used in food
processing, brewing, and detergent production.
o Protease: Degrades proteins into peptides and amino acids, widely
used in the detergent industry for removing protein-based stains
and in food processing.
o Lipase: Hydrolyzes fats into glycerol and fatty acids, used in the
dairy industry for flavor enhancement and in biodiesel production.
o Cellulase: Breaks down cellulose into glucose, used in textile, paper,
and biofuel industries for fabric treatment, pulp processing, and
biomass conversion to bioethanol.
1.4. Antibiotic Production:
 Overview: Antibiotics are secondary metabolites produced by
microbes, especially soil-dwelling bacteria and fungi, to inhibit the
growth of other microorganisms.
 Examples:
o Penicillin: Produced by the fungus Penicillium notatum, penicillin is
one of the first antibiotics discovered and widely used to treat
bacterial infections.
o Streptomycin: Produced by Streptomyces griseus, it is used to treat
tuberculosis and other bacterial diseases.
o Tetracycline: Another antibiotic produced by Streptomyces species,
effective against a broad range of bacterial infections.
 Production Process:
o Fermentation: Large-scale antibiotic production involves
fermentation, where microorganisms are cultured under controlled
conditions to maximize antibiotic yield.
o Downstream Processing: The antibiotic is purified through a series
of steps, including extraction, crystallization, and drying, before it is
formulated into the final product.
1.5. Bioremediation:
 Definition: The use of microorganisms to degrade or detoxify
environmental contaminants such as oil spills, heavy metals, and
pesticides.
 Mechanism:
o Microbial Metabolism: Microorganisms metabolize pollutants by
using them as a source of carbon and energy, breaking them down
into less harmful substances.
o Types of Bioremediation:
 In Situ Bioremediation: Treatment of the contaminated site without
removal of the polluted material. This can involve the addition of
nutrients or oxygen to enhance microbial activity.
 Ex Situ Bioremediation: Removal of the contaminated material
(e.g., soil or water) to a treatment facility where microbial
degradation occurs.
 Applications:
o Oil Spill Cleanup: Microbes such as Alcanivorax species break down
hydrocarbons in oil, helping to clean up marine oil spills.
o Heavy Metal Detoxification: Certain bacteria can transform toxic
metals (e.g., mercury, chromium) into less toxic forms through
reduction reactions.