TUTORIAL QUESTIONS AND ANSWERS

(PRINCIPLES OF BIOTECHNOLOGY MCB 309)

1. Discuss the multifaceted applications of biotechnology across diverse sectors, hence,
highlight the ethical and social considerations associated with biotechnological
advancements and explore future directions.
Answer

 Pharmaceuticals: Developing and producing drugs, vaccines, and therapeutic

proteins.
 Gene Therapy: Treating genetic disorders by introducing or correcting genes.
 Personalized Medicine: Tailoring medical treatment to an individual's genetic
makeup.
 Diagnostics: Creating advanced diagnostic tools and tests.
 Tissue Engineering: Growing artificial organs and tissues for transplantation.
 Gene Therapy: Correcting or replacing defective genes to treat genetic disorders, ,
also boost immune system
 Diagnosis: DNA-based diagnostic tests for diseases (e.g., PCR-based tests for
COVID-19).
 Genetically improvement of microorganisms for the production of pharmaceutical
products.
 Microbiologically based production of human insulin and interferons
 Genetically Modified Crops (GMOs): Creating crops with improved yield,
resistance to pests and diseases, and nutritional content.
 Crop Protection: Developing biopesticides and bioherbicides.
 Animal Biotechnology: Improving livestock breeding and production.

 Reduce post harvest loss

 Increase the efficiency of mineral usage by plants
 Increae nutritional values of foods e.g Vit A rice

 Biofuels: Producing biofuels like ethanol and biodiesel.

 Bioplastics: Creating environmentally friendly plastics.
 Enzyme Production: Developing enzymes for various industrial processes.
 Conservation Biology: Studying and conserving endangered species.
 Ecological Restoration: Using biotechnology to restore damaged ecosystems.

 Wastewater Treatment: Employing bioremediation in sewage treatment.

 Bioremediation: Using microorganisms to clean up pollution.
 Fermentation: Producing food and beverages such as cheese, yogurt, beer, and wine.
 Food Safety: Developing methods for food safety testing.
 Food Preservation: Extending the shelf life of food products.

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  Monoclonal Antibodies: Producing antibodies for therapeutic use.
 Vaccines: Developing and producing vaccines against various diseases.
 Stem Cell Therapy: Using stem cells for regenerative medicine.
 Biopharming: Production and use of transgenic plants and animals genetically
engineered to produce pharmaceutical substances for use in humans or animals.
 Genomic Research: Studying genes and genomes to understand genetics and
evolution.
 Proteomics: Analyzing proteins and their functions.
 Transgenics: Creating organisms with foreign genes for research purposes.
 DNA Analysis: Using DNA profiling for crime scene investigations and paternity
testing.
 Forensic Entomology: Studying insects to determine time of death.
 Bioinformatics: Developing software and databases to analyze biological data, such
as DNA sequences and protein structures.
 Biotechnology in Energy: Exploring ways to use biological processes for renewable
energy production, such as microbial fuel cells. Bioenergy & Biofuel
 Bioethanol Production: Using microorganisms to convert biomass into ethanol as a
biofuel.
 Biotechnology in Space: Researching biotechnological solutions for space
exploration, including life support systems and food production on spacecraft.
 Human Genome Project: This is the determination of the complete sequences of
nucleotide base pairs that make-up human DNA and all the genes it contains. Applications
include.

Ethical and Social Considerations

 Genetic Privacy: Concerns about the use and protection of genetic information.
 Biosecurity: Safeguarding biotechnology from misuse or bio-terrorism.
 Regulation: Ensuring the safe and ethical use of biotechnology through regulations
and guidelines.

5. Future Directions

 Synthetic Biology: Designing and constructing biological systems for specific

purposes.
 CRISPR-Cas9: Precise genome editing with numerous potential applications.
 Biotechnology in Space: Advancements in biotechnology for space exploration and
colonization.

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Question 2: Describe the four phases of the microbial growth curve, highlighting their key

characteristics, factors influencing each phase, and their industrial significance.

Answer

Log Phase

 When microorganisms are introduced into fresh culture medium, usually no

immediate increase in cell number occurs, so this period is called the lag phase

 there is no net increase in mass, the cell is synthesizing new components.

 A lag phase prior to the start of cell division can be necessary for a variety of reasons.

The cells may be old and depleted of ATP, essential cofactors, and ribosomes; these

must be synthesized before growth can begin.

 The medium may be different from the one the microorganism was growing in

previously. Here new enzymes would be needed to use different nutrients.

 Possibly the microorganisms have been injured and require time to recover.

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  This phase may be quite long if the inoculum is from an old culture or one that has

been refrigerated. Inoculation of a culture into a chemically different medium also

results in a longer lag phase.

 On the other hand, when a young, vigorously growing exponential phase culture is

transferred to fresh medium of the same composition, the lag phase will be short or

absent.

Exponential growth

 During the exponential or log phase, microorganisms are growing and dividing at the

maximal rate possible given their genetic potential, the nature of the medium, and the

conditions under which they are growing.

 The microorganisms are dividing and doubling in number at regular intervals.

 The population is most uniform in terms of chemical and physiological properties

during this phase; therefore exponential phase cultures are usually used in

biochemical and physiological studies.

Stationary phase

 Because this is a closed system, eventually population growth ceases and the growth

curve becomes horizontal.

 This stationary phase usually is attained by bacteria at a population level of around

109 cells per ml.

 Microbial populations enter the stationary phase due to nutrient limitation;

 If an essential nutrient is severely depleted, population growth will slow.

 Aerobic organisms often are limited by O2 availability.

 May also cease due to the accumulation of toxic waste products.

 Finally, there is some evidence that growth may cease when a critical population

level is reached.

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  Procaryotes have evolved a number of strategies to survive starvation,which are

morphological changes gene expression and physiology.

 Starving bacteria frequently produce a variety of starvation proteins, which make

the cell much more resistant to damage in a variety of ways.

Death phase

 For many years, the decline in viable cells following stationary cells was described

simply as the “death phase.”

 It was assumed that detrimental environmental changes like nutrient deprivation and

the buildup of toxic wastes caused irreparable harm resulting in loss of viability

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KINETICS OF MICROBIAL GROWTH

1. Growth Rate Equation

𝑑𝑋
= µX
𝑑𝑡

𝑑𝑋
 This equation states that the rate of microbial growth ( 𝑑𝑡 ) is directly proportional to
the current cell concentration (X), where μ is the specific growth rate.

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2. Integration of the Growth Rate Equation
𝑑𝑋
∫ = ∫ 𝜇𝑑𝑡
𝑋

 Separating the variables allows us to integrate both sides, leading to an equation that
describes how the population grows over time.

lnX = μt + C

 After integration, we introduce an integration constant C.

3. Applying Initial Conditions

At t = 0, let X = 𝑋𝑜 , so:

ln𝑋𝑜 = C

 Substituting this into the previous equation:

Ln 𝑋𝑡 = ln 𝑋𝑜 + μt

 Rearranging:

𝑋𝑡 = 𝑋0 𝑒 𝜇𝑡

 This shows that microbial growth follows an exponential pattern during the log
phase.

4. Conversion to Logarithm Base 10

𝜇
log𝑋𝑡 = log𝑋𝑂 + 2.303 𝑡

 Since lnA = 2.303 logA, we convert natural logarithms to base 10 logarithms for
practical use in microbiology.
𝜇.
 A plot of log𝑿𝒕 versus t gives a straight line, with the slope equal to 2.303

5. Generation (Doubling) Time

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  µ𝑡𝑑 = ln 2
 The generation time (𝑡𝑑 ) is the time taken for the population to double in number.
 Since ln2 ≈ 0.693 approx 0.693; ln2 ≈ 0.693, we solve for 𝑡𝑑 :

𝐥𝐧 𝟐 0.693
𝑡𝑑 = =
µ 𝜇

 This equation helps in determining how quickly a microbial culture doubles under
given conditions.

Key Takeaways:

1. Microbial growth follows an exponential pattern where the population doubles

after every generation.
2. The specific growth rate (μ) determines the speed of growth and varies based on
environmental conditions.
3. The generation time (𝑡𝑑 ) helps estimate how fast microorganisms divide in a
given medium.

Question:

A bacterial culture is grown in a nutrient-rich medium, and its growth is monitored using
optical density (OD) at 600 nm over time. The following data is recorded:

Time (t) in hours Optical Density (OD600)

0 0.05
2 0.12
4 0.28
6 0.55
8 1.05
10 2.10

Tasks:

a) Convert the optical density values to log10(OD600) (5 marks)

b) Plot a graph of log10(OD600) (Y-axis) versus time (t) (X-axis). (5 marks)

c) Determine the specific growth rate (μ) from the slope of the straight-line graph using the
equation:
𝜇
Slope = 2.303

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(5 marks)

d) Calculate the generation time (doubling time, ) using the equation:

0.693
𝑡𝑑 = (5 marks)
𝜇

What are the features of a fermenter, and what factors should be considered when designing
and constructing one?
With the appropriate Mathematical derivatives, describe mathematics and kinetics of
microbial growth.

Highlight the Features of a fermenter

1. It must maintain a contamination-free environment while providing optimal

conditions for maximal product production.

2. Fermentors are also known as bioreactors, and they are essential in biotechnology and

industrial microbiology.

3. Fermentors can be categorized as liquid (submerged) or solid state (surface), with

submerged fermentors being more commonly used in industry.

4. Pilot fermentors, which fall between these size extremes, are also used.

5. Typically, only about 75% of the total volume in a fermentor is utilized for actual

fermentation, with the rest reserved for foam and exhaust gases.

6. The most commonly used type of fermentor is the Aerated Stirred Tank Batch

Fermentor.

Factors to be Considered, in Designing and Constructing a Fermentor

 The vessel must be capable of being operated aseptically for a number of days and

should be reliable in long term operations.

 Adequate aeration and agitation should be provided to meet the metabolic

requirements of microorganisms.

 Power consumption should be as low as possible.

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 It must have a system of temperature control.

 It must have a system of pH control.

 Sampling facilities should be provided.

 Evaporation losses from the fermenter should not be excessive.

 The vessel should be designed to require the minimal use of labour in operation

cleaning, harvesting and maintenance.

 The vessel should be suitable for a range of processes.

 It should have smooth internal surface.

 The cheapest material which enables satisfactory result to be achieved should be used.

 The vessel should be of similar geometry to both smaller and larger vessels in the

pilot plant to facilitate scale-up.

 There should be adequate service provision for industrial parts.

1. Outline the steps involved in Recombinant DNA Technology and discuss its
applications in modern biotechnology.
b. Describe the following processes commonly used in Molecular biology
experiments: a) Transformation b) Electroporation c) Transfection
Steps Involved in Recombinant DNA Technology

 Isolation of DNA - Genomic DNA, cDNA, Chemically Synthesis.

 Fragmentation of the DNA using the enzyme Restriction endonucleases.

 Isolation of the desired DNA fragment.

 Isolation of Vector – E.coli Bactria (Plasmid PBR, PUC etc, Cosmid, Phage, YAC,

BAC etc).

 Amplification of the gene of interest.

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  Ligation of the DNA fragment into a suitable vector by the enzyme DNA ligases.

 Transfer of DNA into the host cell – transformation, transduction, electroporation,

liposome fusion, etc

 Culturing the host cells on a suitable medium on a large scale.

 Extraction of the desired products.

 Downstream processing of the products.

Application of Recombinant DNA Technology

i. Gene Mapping

ii. Genetic disorder

iii. Monoclonal antibodies Productions

iv. Gene therapy

v. Gene fingerprinting

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 vi. Vaccine Production

vii. Pharmaceutical Products

Importance of Genetically Engineered Crops

 Genetically engineered crops have desirable genes (as of insect/pest resistance, giving

better yield) incorporated in them.

 Genetically modified crops have

 More tolerance to abiotic stresses such as cold, drought, salinity, heat, etc.

 Insect/pest resistance

 Reduced post-harvest losses

 Efficient mineral usage by plants

 enhanced nutritional value (e.g., Vitamin A rich rice)

State general features of cloning vectors and host bacteria

Answer

Features of cloning vector

1. Origin sequence (ori) required for replication.

2. Selectable trait that enables E. coli that carry the plasmid to be separated from

E. coli that do not (e.g., antibiotic resistance, grow cells on antibiotic; only

those cells with the antibiotic resistance grow in colony).

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 3. Unique restriction site such that an enzyme cuts the plasmid DNA in only one

place. A fragment of DNA cut with the same enzyme can then be inserted into

the plasmid restriction site.

4. Simple marker that allows you to distinguish plasmids that contain inserts

from those that do not (e.g., lacZ+ gene)

Host Cell (Competent Cells)

A good host should have the following properties:

1. It should be easy to transform.

2. It should not hinder replication of recombinant vector.

3. It should not have restriction and methylase activities.

4. It should be deficient in recombination function so that the introduced recombinant

vector is not altered.

5. It should be easy to grow.

6. The recombinant vector should be easily retrieved from the transformed host.

b. State the types and functions of Enzymes used in recombinant DNA Technology

Answer

 DNA ligase is used for joining DNA molecules.

 Alkaline phosphatase is used for dephosphorylation of the vector i.e. removal of

5’ phosphate to avoid recircularization of the cut vector.

 S1 nuclease is used for cutting single stranded nucleic acids.

 Terminal transferase is used for adding homopolymer tails.

 Reverse transcriptase is used for cDNA synthesis.

 DNA polymerases are used for DNA replication.

 RNase H is used for removal of RNA from RNA/DNA hybrid.

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 1. Suppose that a geneticist discovers a new restriction enzyme in the bacterium
Aeromonas ranidae. This restriction enzyme is the first to be isolated from this
bacterial species. Using the standard convention for abbreviating restriction enzymes,
give this new restriction enzyme a name.

3b. Complete the following palindrome sequences, hence Propose any Recognition and
Cleavage Sites that will give the following cleavage patterns:
5’-?GA?CC-3’
3’-C?TA?G-5’
a. 5’ overhanging b. 3’ overhanging c.. Blunt ends d. 5’ protruding

b. Draw a schematic diagram of colicloning vector pBR 322 and hence explaining how the
labeled parts facilitate cloning into a vector.

1. Origin sequence (ori) required for replication.

2. Selectable trait that enables E. coli that carry the plasmid to be separated from E. coli that

do not (e.g., antibiotic resistance, grow cells on antibiotic; only those cells with the antibiotic

resistance grow in colony).

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3. Unique restriction site such that an enzyme cuts the plasmid DNA in only one place. A

fragment of DNA cut with the same enzyme can then be inserted into the plasmid restriction

site.

4. Simple marker that allows you to distinguish plasmids that contain inserts from those that

do not (e.g., lacZ+ gene

Features of a fermenter

7. It must maintain a contamination-free environment while providing optimal

conditions for maximal product production.

8. Fermentors are also known as bioreactors, and they are essential in biotechnology and

industrial microbiology.

9. Fermentors can be categorized as liquid (submerged) or solid state (surface), with

submerged fermentors being more commonly used in industry.

10. Pilot fermentors, which fall between these size extremes, are also used.

11. Typically, only about 75% of the total volume in a fermentor is utilized for actual

fermentation, with the rest reserved for foam and exhaust gases.

12. The most commonly used type of fermentor is the Aerated Stirred Tank Batch

Fermentor.

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 Fig 1: A fermenter

Construction and design of fermentors are primarily the responsibility of engineers, with

biotechnologists and microbiologists needing a basic understanding to use them effectively.

Factors to be Considered, in Designing and Constructing a Fermentor

 The vessel must be capable of being operated aseptically for a number of days and

should be reliable in long term operations.

 Adequate aeration and agitation should be provided to meet the metabolic

requirements of microorganisms.

 Power consumption should be as low as possible.

 It must have a system of temperature control.

 It must have a system of pH control.

 Sampling facilities should be provided.

 Evaporation losses from the fermenter should not be excessive.

 The vessel should be designed to require the minimal use of labour in operation

cleaning, harvesting and maintenance.

 The vessel should be suitable for a range of processes.

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 It should have smooth internal surface.

 The cheapest material which enables satisfactory result to be achieved should be used.

 The vessel should be of similar geometry to both smaller and larger vessels in the

pilot plant to facilitate scale-up.

 There should be adequate service provision for industrial parts.

Vector is an agent that can carry a DNA fragment into a host cell in which it is capable of
replication.
1) It should be autonomously replicating i.e. it should have ori region.
(2) It should contain at least one selectable marker e. g. gene for antibiotic resistance.
(3) It should have unique restriction enzyme site (only one site for one RE) for different REs
to insert foreign DNA.
(4) It should be preferably small in size for easy handling.
(5) It should have relaxed control of replication so that multiple copies can be obtained.
Cloning vectors are
a. 4 kb: plasmid.
b. 20 kb: ʎ phage.
c. 35 kb: cosmid.
d. BAC

Question 6: Highlight the features of the following cloning vectors: Plasmid, λ-phage
Cloning and Cosmid.
6b. State enzymes used in rDNA and their functions

Plasmid
Origin sequence (ori) required for replication.
Selectable trait that enables E. coli that carry the plasmid to be separated from
E. coli that do not (e.g., antibiotic resistance, grow cells on antibiotic; only
those cells with the antibiotic resistance grow in colony).
Unique restriction site such that an enzyme cuts the plasmid DNA in only one
place. A fragment of DNA cut with the same enzyme can then be inserted into
the plasmid restriction site.
Simple marker that allows you to distinguish plasmids that contain inserts
from those that do not (e.g., lacZ+ gene)

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 Phage  cloning vectors:

1. Engineered version of bacteriophage  (infects E. coli).

2. Central region of the  chromosome (linear) is cut with a restriction

enzyme and digested DNA is inserted.

3. DNA is packaged in phage heads to form virus particles.

4. Phages with both ends of the  chromosome and a 37-52 kb insert

replicate by infecting E. coli.

5. Phages replicate using E. coli and the lytic cycle .

6. Produces large quantities of 37-52 kb cloned DNA.

7. Like plasmid vectors, large number of restriction sites available; phage 

cloning vectors useful for larger DNA fragments than pUC19 plasmid

vectors.

Cosmid Cloning Vectors:

i. Features of both plasmid and phage cloning vectors.
ii. Do not occur naturally; circular.
iii. Origin (ori) sequence for E. coli.
iv. Selectable marker, e.g. ampR.
v. Restriction sites.
vi. Phage  cos site permits packaging into  phages and introduction to E. coli cells.
vii. Useful for 37-52 kb.
viii. Cosmids have a selectable marker and MCS from plasmids, and cos site from phage

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