CPE

1. What are the three domains of life?

Bacteria, Eukaryota, Archea.
Eukaryote: Cells that contain membrane bound organelles like a nucleus

2. - A light microscope: Light rays pass through the specimen on a slide and are focused by the objective lens
through the eye piece lens
- Electron microscope: Uses beams of electrons (not light) -these have a smaller wavelength than light
beams and therefore has higher resolution
3. Actual size of object = size of image / magnification

4. define the differences between prokaryotes and eukaryotes

Prokaryotes lack a nucleus and membrane-bound organelles, are generally smaller and simpler in structure
than eukaryotes, reproduce asexually and divide through binary fission, and have flagella composed of
flagellin. Eukaryotes have a nucleus and membrane-bound organelles, are generally larger and more complex
in structure, can reproduce both sexually and asexually, divide through mitosis or meiosis, and have flagella
composed of microtubules. These differences reflect their distinct evolutionary pathways.

5. What do cells do?

Metabolism, biosynthesis, transcription and translation.

6. Elemental composition of bacteria

Bacteria are primarily composed of carbon, oxygen, hydrogen, and nitrogen, which make up about 95% of the
dry weight of the cell. Other elements, such as sulfur, phosphorus, potassium, magnesium, calcium, iron, and
trace elements, are also important.

7. Four basic classes of macromolecules: Carbohydrates, Lipids, Proteins, Nucleic Acids.

brief overview of each macromolecule:

 Carbohydrates: These are made up of simple sugars, and they are an important source of energy for cells.
- Monosaccharides and disaccharides are simple sugars that provide a source of energy for cells. They are
broken down during cellular respiration to produce ATP, which is used for various cellular processes.
- Polysaccharides: made of hundreds or even thousands of monosaccharides linked together in long chains.
Mostly insoluble so great for energy storage (e.g. starch) or forming strong structures (e.g. cellulose).
 Lipids: These include fats, oils, and waxes, and they serve as a source of energy and as a structural component
of cell membranes. Lipids also play important signaling roles within cells and between cells.
 Proteins: These are made up of chains of amino acids, and they perform a wide range of functions in cells.
Proteins serve as enzymes that catalyze chemical reactions, as structural components of cells, and as signaling
molecules.
- 5 major categories of proteins – Structural proteins – glycoproteins, collagen, keratin – Catalytic proteins -
enzymes – Transport proteins – haemoglobin – Regulatory proteins – hormones – Protective proteins –
antibodies
 Nucleic acids: These include DNA and RNA, and they are responsible for storing and transmitting genetic
information. DNA is the blueprint for making proteins, while RNA plays a central role in protein synthesis.
- DNA is double stranded-A forms hydrogen bonds with T, and G form hydrogen bonds with C
- RNA uses Uracil (U) not Thymine (T)
 Metabolism
The sum of the chemical reactions that take place within each cell of a living organism and that provide
energy for vital processes and for synthesizing new organic material.
• 2 types: -catabolism (breaking down organic matter to produce energy via respiration)
-anabolism (using energy to make macromolecules like proteins)

Respiration
• Cells release energy from glucose in a process called respiration • The energy released is used to power
cellular processes • There are two types -aerobic respiration and anaerobic respiration • Aerobic respiration
produces CO2 and H2O and releases energy
In respiration the energy released from glucose is used to make Adenosine triphosphate (ATP) a nucleotide
whose reactivity resides in its terminal phosphate groups.

ATP
Adenosine triphosphate (ATP) is a molecule that serves as the primary source of energy for cellular processes
in living organisms. ATP is produced through cellular respiration and photosynthesis, and it can be used to
power cellular processes such as muscle contraction, protein synthesis, and active transport across cell
membranes. ATP is made up of a nucleotide base (adenine), a sugar (ribose), and three phosphate groups, and
it releases energy when its terminal phosphate group is hydrolyzed.

Oxidation/Reduction in respiration:

- Cellular respiration involves a series of oxidation-reduction (redox) reactions that ultimately produce
ATP, the primary energy currency of cells. During respiration, glucose is oxidized to produce CO2 and
H2O, while oxygen is reduced to form H2O. Electrons are transferred from glucose to oxygen through a
series of redox reactions that release energy, which is used to generate ATP.
- • Glucose is an energy rich food due to lots of C-H bonds.
- • Energy is released when it is oxidised as electrons are relocated closer to oxygen.
- • Slow energy release avoids loss of it in the form of heat
- • Cells release the energy slowly using enzymecatalysed steps (as hydrogen are stripped away) in
glycolysis and citric acid cycle.

 Organisms extract energy from food in stages

- Food is digested- CHO to sugars, proteins to amino acids, fats to fatty acids
- Simpler compounds undergo catabolism- energy stored in chemical bonds is used to power ATP production

 There are 3 main stages in respiration 1) Glycolysis 2) Krebs cycle (citric acid cycle) 3) Oxidative
phosphorylation

1. Two stages in glycolysis

Phosphorylation of glucose (adding 2 phosphates from 2 ATP) to make 2 molecules of triose phosphate Oxidation
of triose phosphate to make 2 pyruvate molecules, NAD collects H+ ions, 4 ATP’s made
What is the link reaction?
The link reaction, also known as the pyruvate dehydrogenase complex reaction, is a key step in cellular respiration. It
occurs between glycolysis and the Krebs cycle and involves the conversion of pyruvate to acetyl-CoA. The reaction
generates NADH and CO2 and prepares the acetyl-CoA to enter the Krebs cycle.
2. citric acid cycle / Krebs cycle
The Krebs cycle, also known as the citric acid cycle, is a metabolic pathway that occurs in the mitochondria of
eukaryotic cells. It is the second stage of cellular respiration and serves to complete the oxidation of glucose
by breaking down acetyl-CoA to produce CO2, ATP, and electron carriers NADH and FADH2. The cycle
involves a series of enzymatic reactions, including the formation of citrate from acetyl-CoA, the release of
CO2 and energy-rich electrons, and the regeneration of oxaloacetate. The electron carriers NADH and
FADH2 are then used in the electron transport chain to generate ATP through oxidative phosphorylation.

3. Oxidative phosphorylation
Oxidative phosphorylation is the final stage of cellular respiration, occurring in the mitochondria of
eukaryotic cells. It involves the transfer of electrons from electron carriers NADH and FADH2 to a series of
proteins called the electron transport chain, located in the inner mitochondrial membrane. The electron
transfer generates a proton gradient across the membrane, which is used by the ATP synthase enzyme to
generate ATP from ADP and inorganic phosphate through a process called chemiosmosis. Oxidative
phosphorylation is responsible for generating most of the ATP produced in cellular respiration, making it a
critical process for sustaining cellular energy metabolism.

METABOLIC ENGINEERINNG

What is metabolic engineering?

Directed improvement of product formation or cellular properties through the modification of specific
biochemical reaction(s) or the introduction of new one(s) with the use of recombinant DNA technology.

What is the difference between metabolic engineering and biochemical engineering?

Metabolic engineering focuses on modifying metabolic pathways within cells to optimize them for specific
functions or to produce desired products. Biochemical engineering, on the other hand, involves the design and
optimization of processes that use living cells or biological molecules to produce valuable products. It
typically involves the development of bioreactors and other tools for scaling up production.

How can we increase production?

There are several strategies that can be used to increase production in metabolic engineering:
- Gene overexpression: Increasing the expression of genes involved in the production pathway can
increase the amount of the desired product.
- Metabolic flux analysis: Analyzing and optimizing the metabolic flux, or the rate of reactions in the
pathway, can improve production efficiency.
- Substrate and cofactor engineering: Modifying the substrates and cofactors involved in the pathway
can improve production efficiency.
- Synthetic biology: Using synthetic biology tools, such as CRISPR/Cas9 genome editing, can enable
the creation of new metabolic pathways or the optimization of existing ones.
- Process optimization: Improving the conditions in which the production process takes place, such as
pH, temperature, and aeration, can increase production efficiency.

Mechanisms of metabolic regulation:

- Modulation of activity of enzymes (inhibition, feedback inhibition and activation, phosphorylation or other
post-translational modifications)
- Concentration of enzymes (synthesis rate, protein degradation)
Measuring flux

What if you need to increase biomass to make more product?

To increase biomass for the purpose of producing a desired product in metabolic engineering, strategies such as
improving nutrient availability, optimizing growth conditions, increasing expression of biomass-associated genes,
reducing by-product formation, and using growth-promoting compounds can be employed. However, optimizing the
metabolic pathway and fluxes towards the desired product is also essential for successful metabolic engineering.

what is metabolic network modelling in 60 words

Metabolic network modeling is a computational approach used in systems biology to study the behavior of metabolic
pathways. It involves constructing mathematical models of the interconnected metabolic reactions that occur within a
cell or organism. These models can be used to predict metabolic fluxes, identify potential drug targets, and optimize
metabolic engineering strategies. Metabolic network modeling typically involves the use of algorithms and software
tools to simulate and analyze the complex behavior of metabolic systems.
 DNA
What is DNA?
DNA, or deoxyribonucleic acid, is a molecule that contains the genetic information necessary for the development,
functioning, and reproduction of all living organisms. It is composed of four nucleotide bases, adenine, thymine,
cytosine, and guanine, and forms a double helix structure that is capable of replication and transmission from one
generation to the next. Each nucleotide is made from a pentose sugar (deoxyribose), a phosphate group and a
nitrogenous base

What is the central dogma of biology?

The central dogma of biology is the fundamental principle that describes the flow of genetic information in living
organisms. It states that genetic information flows from DNA to RNA (transcription), and then from RNA to protein
(translation). This process is unidirectional, meaning that protein cannot be converted back into RNA or DNA.

Structure of DNA;
The structure of DNA (deoxyribonucleic acid) is a double-stranded helix composed of four nucleotide bases: adenine
(A), thymine (T), cytosine (C), and guanine (G). The nucleotides are linked together by phosphodiester bonds between
the sugar and phosphate groups, forming a backbone for each strand. The two strands are held together by hydrogen
bonds between complementary base pairs, A-T and C-G. The double helix structure of DNA is essential for its
function as a carrier of genetic information, as it allows for accurate replication and transmission of genetic material
from one generation to the next.

Synthetic Biology in 3 points:

- To design and construct biological parts and devices to our own specification
- The devices are based on specific parts of natural organisms which biologists have identified and extracted.
- These ‘parts’ would be incorporated into existing living organisms to redesign them to carry out new
functions useful to us

What is a standardised part?

In synthetic biology, a standardized part refers to a DNA sequence that encodes a specific biological function and
is characterized and documented in a standardized format, enabling it to be easily integrated with other genetic
parts and modules.
 INDUSTRIAL MICROBIOLOGY
Growth Kinetics N = N02n
N = final cell number, N0 = initial cell number, n = number of generations during exponential growth.

The four phases of microbial growth in batch culture are:

1. Lag phase: A period of low or no growth, where cells are adjusting to the new environment and synthesizing
necessary components for cell division.
2. Exponential (log) phase: A period of rapid growth, where cells are dividing at their maximum rate and the
population is increasing exponentially.
3. Stationary phase: A period of no net growth, where the growth rate slows and the number of viable cells
reaches a plateau due to nutrient depletion and accumulation of waste products.
4. Death phase: A period of decline, where the number of viable cells decreases as the cells enter a state of
dormancy or die off due to the accumulation of toxic waste products and nutrient depletion.

The Monod model is a mathematical model

that describes the relationship between the
growth rate of microorganisms, substrate
concentration, and the saturation constant
in a batch culture. It is used to optimize
bioprocesses and design bioreactors but has
limitations.

Single use bioreactors:

Eliminates need for extensive cleaning and steam
sterilization between batches Can introduce
environmental impacts related to the manufacturing,
use, and disposal of consumable materials
MICROBIOLOGY III
What are the disadvantages of current host cell systems?
Costly media (e.g. mammalian cell lines for protein therapeutics, carbon in plastics) • Waste- single use bioreactors,
plastics etc. • Environmental footprint
What is bioprocessing?
Bioprocessing defined as activities that enable design, development and production of biological medicines both in
bulk and final dosage form. Several stages including cell line development, cell banking fermentation processes,
recovery and purification processes, chemical modification, formulation process, dosage form, storage and stability,
analytical methods for in-process, final product and stability testing.

What is transcription?
Transcription is the process by which genetic information encoded in DNA is copied into RNA. It is the first step in
gene expression, where the genetic code is transferred from the DNA molecule to the RNA molecule. The process
involves the binding of RNA polymerase to a specific region of the DNA called the promoter, followed by the
synthesis of a complementary RNA molecule. The RNA molecule is then further processed to produce functional gene
products such as proteins.
DNA transcription produces a single-stranded RNA molecule that is complementary to one strand of DNA.

What is translation?
Translation is the process by which the genetic information carried by messenger RNA (mRNA) is converted into a
sequence of amino acids to form a protein. It occurs in the ribosomes, where transfer RNA (tRNA) molecules bring
specific amino acids and align them according to the mRNA codons. The ribosome catalyzes the formation of peptide
bonds between the amino acids, resulting in the synthesis of a functional protein.
The process of interpreting DNA to make a protein involves two main steps: transcription and translation.
Transcription:
1. Initiation: RNA polymerase binds to a specific region of DNA called the promoter. This signals the start of
transcription.
2. Elongation: RNA polymerase unwinds the DNA double helix and synthesizes a complementary RNA
molecule using the DNA template strand. Adenine (A) in DNA is replaced with uracil (U) in RNA.
3. Termination: Transcription continues until a termination signal is reached, causing RNA polymerase to
dissociate from the DNA template. This results in the formation of a newly synthesized mRNA molecule.
Translation:
1. Initiation: The mRNA molecule binds to the small ribosomal subunit, and the ribosome scans the mRNA until
it reaches the start codon (usually AUG).
2. Elongation: tRNA molecules carrying specific amino acids enter the ribosome, pairing their anticodons with
the codons on the mRNA. Peptide bonds form between adjacent amino acids, forming a growing polypeptide
chain.
3. Termination: When a stop codon (UAA, UAG, or UGA) is reached, a release factor binds to the ribosome,
causing the polypeptide chain to be released. The ribosome and mRNA dissociate.
After translation, the polypeptide chain may undergo post-translational modifications to fold into its functional three-
dimensional structure and may be targeted to specific cellular compartments for its proper functioning.

How does the order of nucleotides in a DNA molecule encode the information that specifies the order of amino
acids in a polypeptide?
The order of nucleotides in a DNA molecule encodes the information for the order of amino acids in a polypeptide
through the genetic code. During transcription, DNA is transcribed into mRNA, which carries the genetic code to the
ribosomes. During translation, the ribosomes read the mRNA codons and match them with specific amino acids,
resulting in the synthesis of a polypeptide with a specific amino acid sequence.
Are the codons in a gene just next to each other or punctuated with non-transcribed nucleotides?
In most cases, the codons in a gene are contiguous, meaning they are located next to each other without non-
transcribed nucleotides interrupting the codon sequence. The coding region of a gene consists of a series of codons
that specify the order of amino acids in the corresponding protein. However, there are certain non-coding regions
within genes, such as introns in eukaryotic genes, that are transcribed but not translated into protein. These non-coding
regions are generally removed during post-transcriptional processing, leaving only the contiguous codons in the final
mRNA molecule for translation.

What is gene regulation?

Gene regulation refers to the mechanisms by which cells control the expression of genes. It involves a complex set of
processes that determine when and to what extent a gene is transcribed into mRNA and translated into a protein. Gene
regulation allows cells to respond to internal and external cues, maintain homeostasis, and ensure appropriate gene
expression patterns during development, differentiation, and various physiological processes.

What is a catalyst? Catalysts are substances that increase or decrease the rate of a chemical reaction but remain
unchanged.
What is a biocatalyst? Enzymes. Proteins that increase the rate of chemical reactions converting substrate into
product. Biocatalysis is the use of natural catalysts such as protein enzymes to perform chemical transformations on
organic compounds.

What are Enzymes? • Globular proteins with precise 3D structures • Chain of amino acids (primary structure) folds
and twists into secondary and tertiary structure and (quaternary structure) • Biological Catalysts – Increase the rate
of a chemical reaction without undergoing permanent chemical change themselves – Enzymes do not affect the
reaction equilibrium - only the rate of reaction
Major Properties of Enzymes
1. High Catalytic Power – 109 to 1012 fold increase compared to nonenzymatic activity
2. Specificity - Substrate and type of reaction
3. Tend to operate under mild conditions, pH, temp, - pressure (Topt = 20-37oC)
4. Regulation – Activity is regulated by cofactors
• Name enzymes by adding suffix –ase e.g. lactase
• Holoenzyme = apoenzyme + cofactor
• Apoenzyme – Protein Part – Inactive Form
• Cofactor – Non-protein part
1) Metal ions e.g K+, Mg2+
2) Coenzyme- organic compounds - often derivatives of B group vitamins - Loosely attached to protein
3) Prosthetic group- organic compounds- firmly attached to protein

Classification of Enzymes

• Oxidoreductases – Catalyse oxidation (loss of electrons) and reduction (gain of electrons) reactions

• Transferases – Group transfer reactions – e.g. CH3 (methyl) groups or phosphates (kinase)

• Hydrolases – Hydrolytic reactions – Break open compounds with the addition of water – e.g. Phosphatase (removes
phosphate)

• Lyases – Elimination reactions – Form double bonds

• Isomerases – intramolecular rearrangements or catalyse conformational changes. – substrate AND product have
the same chemical formula

• Ligases (synthetases) – Joining of two molecules together – Requires energy, usually ATP
Competitive vs non-competitive inhibitors
Competitive inhibitors are molecules that bind to the active site of an enzyme, competing with the substrate for
binding. This inhibits the enzyme's activity by preventing the substrate from binding and forming the product.
Increasing substrate concentration can overcome the inhibition.
Non-competitive inhibitors, on the other hand, bind to a different site on the enzyme called the allosteric site. This
binding induces a conformational change in the enzyme that affects its active site, reducing its catalytic activity. Non-
competitive inhibitors are not overcome by increasing substrate concentration.
Both competitive and non-competitive inhibitors regulate enzyme activity and can be used as tools in drug
development and understanding enzyme kinetics.

The enzyme unit

• a unit of enzyme's catalytic activity (U)

• U (μmol/min) is defined as the amount of the

enzyme that catalyses the conversion of one micromole of substrate per minute under the specified conditions of
the assay method.