Molecular Systems Biology (2023)

Topics for examination

Each module is worth 22 points. Each module will contain 5 multiple choice questions and
2 open questions.

Module 1

Study materials (uploaded in Moodle):

Uri Alon, „An Introduction to Systems Biology“ 2nd edition – Chapters 9, 12, Appendix D.
Lecture slides and papers uploaded next to them

Topics:
1. Name and explain four potential sources of molecular noise in cellular systems.

The inherent stochasticity of biochemical processes that are dependent on infrequent

molecular events involving small numbers of molecules;
• (ii) variation in gene expression owing to differences in the internal states of a population of
cells, either from predictable processes such as cell cycle progression or from a random
process such as partitioning of mitochondria during cell division;
• (iii) subtle environmental differences, such as morphogen gradients in multicellular
development;
• (iv) ongoing genetic mutation, either random or directed.

2. Name and explain two main types of molecular noise.

Intrinsic and extrinsic noises

Intrinsic noise – due to stochastic events during gene expression
• Extrinsic noise – due to any existing cellular heterogeneity that affects gene expression or
to stochastic events in upstream signal transduction

3. Explain a method to study the extrinsic and intrinsic noise separately. Describe
an experimental setup and possible results.

CFP and YFP values for cell (solid circles) during a time course of PHO5 induction by
phosphate starvation. Populations from different time points are indicated. Extrinsic
noise is manifested as scatter along the diagonal and intrinsic noise as scatter
perpendicular to the diagonal

4. How do the frequencies of the extrinsic noise, intrinsic noise and cell
cycle fluctuations differ (transcriptional noise)?
5. Name and explain four possible ways to control intrinsic noise in gene expression.

Infrequent transcription followed by efficient translation results in high intrinsic noise in

protein levels (left); frequent transcription and inefficient translation results in low intrinsic
noise (right)
Negative feedback, as when a transcription factor represses its own transcription (right),
results in decreased noise relative to a linear pathway (left).
 Infrequent promoter transitions between inactive and active states followed by efficient
transcription result in high intrinsic noise in mRNA levels (left); frequent promoter transitions
followed by inefficient transcription result in low intrinsic noise (right).
Increases in gene copy number through polyploidy (top right) or gene duplication (bottom
right) result in decreased intrinsic noise relative to a single gene copy (left).

6. Explain three concepts of gene expression noise.

• Bursts: Proteins do not trickle out at a uniform rate, but rather are produced in stochastic
bursts. This is both because each individual messenger RNA is typically translated many times
to produce many proteins and also because the gene’s promoter can stochastically switch
between long-lived ‘off’ and ‘on’ states, resulting in bursts of mRNA production amplified to
generate corresponding protein bursts.
• Time-averaging: When the protein lifetime is longer than the interval between protein
production bursts (as it often is), the accumulation of proteins over time tends to average
out the variability generated by bursty expression, effectively buffering the protein
concentration.
Propagation: Rates of gene expression are influenced directly by the levels and states of
transcription factors and other upstream components that are themselves subject to
bursting and time averaging. As a result, fluctuations in the expression of one gene
propagate to generate fluctuations in downstream genes

7. Describe three functional roles of noise in probabilistic differentiations.

1) Stochastic state-switching systems switch between metastable states and are often based
on positive feedback loops.
2) Noise-triggered excitable differentiation systems allow cells to probabilistically enter a
state, but return to the original state after a defined time. These systems use a combination
of positive and negative feedback loops.
3) In procrastinating differentiation systems, individual cells gradually and variably build up
the level of a key regulator to generate a broad distribution of delays before committing to a
new, markedly different fate

8. How does the phenotypic noise change during the evolution (during the adaptation
to new environments and when the selection becomes stabilized).

Phenotypic noise increases during periods of adaptation to new environments, followed

by reduction of noise when selection becomes stabilizing.

9. What is the fundamental difference between fine tuned and robust circuits.

• Biological circuits have robust designs such that their essential function is nearly
independent of biochemical parameters that tend to vary from cell to cell.
• A property of a circuit is robust with respect to certain parameters. Robustness is never
absolute: it is a relative measure. Properties that are not robust are fine-tuned
• The principle of robustness can help us to rule out a large family of plausible mechanisms
of the studied circuit.

10. Explain the general meaning of bacterial chemotaxis.

Bacterial chemotaxis – a process in which bacteria sense and move along gradients of specific
chemicals.
 Chemotaxis enables bacteria to sense their immediate environment and quickly adapt to
changes in it. The ability to sense changes in the environment and quickly respond to them is
essential to the cell's life.

11. Explain the meaning of the principle of exact adaptation in the context of bacterial
chemotaxis.

Exact adaptation means that the steady-state tumbling-frequency does not depend on
the level of attractant
Exact adaptation depends on tuning between the reduction in CheB velocity and the
reduction in activity per methylated receptor upon attractant binding, so that the activity
per methylated receptor times the number of methylated receptors returns to the pre-
stimulus level.

12. Explain the chemotaxis protein network and the role of CheY, CheB, CheR in it.

the chemotaxis protein network in bacteria involves a coordinated interplay between

proteins like CheY, CheB, and CheR. CheY acts as a response regulator that influences
flagellar motor behavior, while CheB and CheR are involved in the adaptation phase by
modulating the methylation state of MCPs, allowing the bacterium to sense and respond to
changes in its chemical environment effectively. This complex regulatory system enables
bacteria to navigate their surroundings and locate optimal conditions for survival and
growth.

13. Explain the Barkai-Liebler robust mechanism of adaptation in bacterial chemotaxis.

Un-methylated receptors are methylated by CheR at a constant rate. Methylated receptors

(marked with m) transit rapidly between active (marked with a star) and inactive states.
Attractant binding increases the rate to become inactive, whereas repellents increase the
rate to become active. De-methylation is due to CheB, which acts only on the active
methylated receptors. The active receptors catalyze the phosphorylation of CheY, leading to
tumbles. Exact adaptation occurs when the concentration of active receptors adjusts itself so
that the de-methylation rate is equal to the constant methylation rate.

14. Explain the meaning of robust design of biological circuits.

A property of a biological circuit is robust if it is nearly independent of the biochemical

parameters that vary unavoidably from cell to cell or system to system.

15. Name two alternative general ways to achieve molecular patterning.

Patterning is a process that generates spatially nonuniform gene expression patterns or, in a
wider sense, spatially heterogeneous cellular responses. There are two ways to achieve
patterning: 1) one that is spontaneous, resulting from the intrinsic instability of particular
reaction diffusion systems, as represented by Turing patterns. 2) Another that is more
programmatic, where patterns are generated through the interpretation of morphogen
gradients by gene regulatory networks (GRNs), including those involving transcriptional
regulation and protein– protein interactions.

16. Explain three diferent ways to combine FFLs and FBLs in pattern formation and their
 outcomes.

Series of iFFLs (incoherent Feed-Forward Loops): Doubles the stripe number. Parallel iFFLs:
Adds a stripe. Combination of iFFL and pFBL (positive Feedback Loop): Generates a single
stripe with a sharp boundary.

17. Explain the French flag model of morphogenetic gradients in pattern formation.

Morphogen M is produced at a specific point and diffuses into a field of cells. Cells assume
different fates (A, B, C) based on the concentration of M. The model is analogous to a French
flag, where different regions represent different cell fates based on morphogen
concentration thresholds.

18. Write one-dimentional diffusion-degradation equation of the morphogen gradient.

?M/?t = D ?**2M/?**2x**2 – aM
D is the diffusion coefficient, a is the degradation rate, t is time, and x is position along
gradient.

19. How would the self-degradation of the morphogen affect the robustness of
morphogen diffusion profile. Write a diffusion-degradation equation for such a case.
Draw two network motifs that could provide such self degradation.

Self-degradation can enhance robustness.

?M/?t = D ?**2M/?**2x**2 – aM**2
D is the diffusion coefficient, a is the degradation rate, t is time, and x is position along
gradient.
Network motifs providing self-degradation:
(a) Morphogen binds receptor, activates signaling pathways that increase receptor expression,
and undergoes endocytosis for degradation.
(b) Morphogen binds receptor, activates pathways repressing receptor expression, and binds
an extracellular protein (protease) that degrades morphogen.

20. Explain the Eldar-Barkai model of robust dorsal patterning in Drosophila embryos
and name the two special conditions related to morphogen and inhibitor that were
predicted via a modeling approach.

Model tested for robustness against changes in gene dosage of morphogen (M), inhibitor
(I), and protease (P).
Special conditions for robustness: (1) Free M cannot diffuse much (DC >> DM), and (2) Free
I is not degraded by P (ac >> aI).
Analytical solution for the steady state:
M(x)=2DI/kx**2

.
Module 2

Study materials (uploaded in Moodle):

Jens Nielsen (2017) Systems Biology of Metabolism

Topics:
1. Describe the difference between top-down and bottom-up systems biology

Top-down Systems Biology: Analyzes high-throughput omics data in an integrative fashion. It

focuses on understanding complex interactions within biological systems using holistic
approaches.
Bottom-up Systems Biology: Assembles detailed models for specific processes (e.g.,
enzymatic reactions) into a model describing the system being studied. It is more reductionist
in nature.

2. Genomics – definition and applications

Definition: Genomics is an interdisciplinary field focusing on the structure, function,

evolution, mapping, and editing of genomes.
Applications: Understanding the complete set of DNA in an organism, studying genes, and
characterizing and quantifying genes for various purposes.

3. Principles of the next-generation sequencing technologies

Illumina Sequencing: Utilizes a workflow involving bridge amplification, reversible terminators,

and fluorescence detection.
PacBio (Single-Molecule, Real-Time DNA Sequencing): Observes the temporal order of
fluorescently labeled nucleotide incorporations during unhindered DNA synthesis.
Ion Torrent: Leverages semiconductor technology for fast, direct detection.
Oxford Nanopore Sequencing: Utilizes nanopores to sequence DNA.

4. Transcriptomics – definition and applications

Definition: Transcriptomics studies an organism's transcriptome, which is the sum of all its RNA
transcripts.
Applications: Analyzing gene expression, detecting novel transcripts, mapping exon/intron
boundaries, studying alternative splicing, and revealing sequence variations.

5. DNA microarrays – working principles and analysis workflow

Working Principles: Microarrays are collections of microscopic DNA spots on a solid surface. They
measure gene expression levels or genotype multiple regions of a genome.
Analysis Workflow: Involves chip preparation, sample preparation, hybridization, and data
acquisition.

6. RNA sequencing – working principles, analysis workflow, and benefits

Working Principles: High-throughput sequencing of cDNA using NGS technologies.

Module 3

Study materials (uploaded in Moodle):

Analysis Workflow: Involves RNA extraction, library preparation, sequencing, and bioinformatic
analysis.
Benefits: Provides information on absolute transcript levels, detects novel transcripts, and allows
the analysis of alternative splicing.

7. Proteomics – definition and applications

Definition: Proteomics studies the entire set of proteins produced by an organism.

Applications: Identifying, determining levels, localizing, and studying post-translational
modifications and interaction partners of proteins.

8. Factors influencing protein levels

Diverse chemical composition of proteins.

Extreme differences in protein abundances.
Post-translational modifications (PTMs).
Interaction partners.

9. Types and regulation of post-translational modifications (PTMs)

Types: Phosphorylation, glycosylation, acetylation, methylation, ubiquitination, etc.

Regulation: PTMs regulate protein function, stability, localization, and interaction with other
molecules.

10. Liquid chromatography working principles

Separates compounds based on their interaction with a stationary phase in a liquid mobile
phase.
Different modes include normal phase, reversed phase, and hydrophilic interaction liquid
chromatography (HILIC).
11. Mass-spectrometry working principles

Ionizes molecules, separates ions based on their mass-to-charge ratio (Mw/z), and detects them.
MS/MS involves fragmentation for sequence determination.

12. Mass-spectrometry-based proteomic analysis workflow

Involves sample preparation, enzymatic digestion, liquid chromatography separation, mass

spectrometry analysis, and data interpretation.

13. Differences between relative and absolute quantification

Relative Quantification: Compares intensity differences between two samples, resulting in

fold-change values.
Absolute Quantification: Compares sample intensity to a known standard, providing absolute
Module 4

Study materials (uploaded in Moodle):

quantity.

14. Metabolomics – definition and applications

Definition: Metabolomics studies the complete set of metabolites in an organism.

Applications: Understanding the phenotype, detecting disease biomarkers, and studying the
effects of drugs and environmental factors.

15. Typical metabolomic analysis workflow

16. Integrative data analysis: qualitative vs. quantitative

Qualitative Integrative Data Analysis (IDA): Involves the visualization of relationships

between different data sources without assigning numerical values. Examples include
heatmaps, dendrograms, and network visualizations. Qualitative IDA is exploratory and helps
identify patterns or clusters within the data.

Quantitative Integrative Data Analysis (IDA): Entails the measurement of relationships

between different data sources using statistical methods or mathematical models. Examples
include correlation analysis, gene-set analysis, and network-based analysis. Quantitative IDA
aims to provide a more rigorous understanding of the relationships and their significance.

17. Gene set analysis and enrichment analysis

Gene Set Analysis (GSA): Involves grouping genes into sets based on biological functions or
pathways. GSA evaluates whether the genes within a set show coordinated changes in
expression or other relevant features. Common methods include Fisher's exact test or
hypergeometric tests.
Enrichment Analysis: Determines whether a predefined set of genes (e.g., a pathway or
functional group) is overrepresented among genes that show significant changes in a
particular condition. It helps identify biological functions or pathways that are enriched with
differentially expressed genes.

18. Visualization tools: heatmaps, principal component analysis, Circos plots

Heatmaps: Visual representation of data where values in a matrix are represented as colors.
Heatmaps are often used to display patterns, such as clustering of samples or genes based on
similarity.

Principal Component Analysis (PCA): A dimensionality reduction technique that transforms

high-dimensional data into a lower-dimensional space while preserving the most significant
variance. It is commonly used for visualizing relationships and clustering in multivariate data.

Circos Plots: Circular visualizations used to display relationships between different entities,
such as genes or genomic regions. Circos plots are often employed in integrative data analysis
to illustrate connections and interactions across multiple layers of data.

These visualization tools aid researchers in gaining insights into complex biological data,
facilitating the interpretation of patterns and relationships within the datasets.
Module 5

Study materials (uploaded in Moodle):

Campbell, Xia, Nielsen - 2017 - The Impact of Systems Biology on Bioprocessing
Liew et al. - 2016 - Gas Fermentation – A Flexible Platform for Commercial Scale Production
of Low Carbon Fuels and Chemicals from Waste
O’Brien, Monk, Palsson - 2015 - Using Genome-scale Models to Predict Biological
Capabilities

Topics:
1. Metabolic modelling: rationale and methods

Metabolic modeling is essential for understanding and predicting cellular behavior. It

involves creating mathematical representations of cellular metabolic pathways to analyze
and simulate the flow of metabolites. Various methods, including kinetic modeling,
stoichiometric modeling, and whole-cell models, are employed to gain insights into cellular
metabolism.

2. Genome-scale stoichiometric metabolic modelling: what and why

Genome-scale stoichiometric metabolic modeling involves representing all metabolic

reactions in an organism, considering their stoichiometry, gene associations, directionality,
and input/output relationships. This comprehensive approach allows for a holistic
understanding of cellular metabolism, aiding in the prediction of cellular phenotypes and the
identification of potential targets for engineering.

3. Main steps for reconstruction of genome-scale stoichiometric models

1-Draft reconstruction
2-Curated reconstruction
3-Genome-scale metabolic model

4. The S matrix in stoichiometric models – what, why, and limitations

The stoichiometric matrix (S matrix) describes the relationships between metabolites and
reactions in a metabolic network. It is crucial for understanding mass balances in the system
and is the foundation for flux balance analysis (FBA). However, the S matrix simplifies the
system by neglecting factors like enzyme kinetics. Enzymes influence rates and kinetics •
Activation energy • Substrate affinity • Rate constants

5. Constraints in stoichiometric models – why and what type

Constraints in stoichiometric models are applied to reflect physiological limitations and real-
world conditions. These constraints can include substrate uptake rates, biomass production
rates, and other biologically relevant parameters. They help in making the model more
realistic and applicable to experimental conditions. Like lower and upper bounds of fluxes,
reaction directionality,

6. Objective function in stoichiometric models – what, why, and limitations

The objective function in stoichiometric models represents the biological goal, often the
maximization of biomass or a specific product. It serves as a basis for optimization algorithms
Module 6

Study materials (uploaded in Moodle):

and helps predict cellular behaviors. However, selecting the appropriate objective function
can be challenging, and it may not fully capture the complexity of cellular objectives.

7. Three assumptions for flux balance analysis

Flux balance analysis (FBA) relies on three fundamental assumptions: completeness of the
reaction network, steady-state conditions (no metabolite accumulation), and the existence of
an organizing principle guiding flux distribution, often assumed to be optimality.

8. Linear optimisation in stoichiometric models – basics and types of solutions

Linear programming is used to solve stoichiometric models, aiming to optimize the objective
function while satisfying the given constraints. Different types of solutions, including unique
solutions, alternative solutions, and degenerate solutions, single solution can arise based on
the model and constraints.

9. Applications of genome-scale metabolic models and more advanced models

Genome-scale metabolic models find applications in diverse fields, including metabolic

engineering, drug discovery, and understanding diseases. More advanced models, such as
whole-cell models, allow for a more comprehensive representation of cellular processes,
enabling a deeper understanding of cellular behavior. Topological enrichment, for context
specific flux distributions, for cell and tissue-specific model building

10. Concept of gas fermentation

Gas fermentation involves using gases such as CO, CO2, and H2 as feedstocks for microbial
growth and product formation. This approach offers advantages in terms of utilizing waste
gases and reducing reliance on traditional carbon sources.

11. Rationale and advantages of gas fermentation

Gas fermentation provides a sustainable and versatile approach to produce fuels and
chemicals. It allows the utilization of waste gases, reduces greenhouse gas emissions, and
offers potential for producing biofuels and biochemicals economically. Even with the
extensive biomass processing required to produce
cellulosic ethanol, the lignin, which can account for up to 40%
of plant biomass does not get converted (Sun and Cheng, 2002;
Abubackar et al., 2011). The use of the biomass in its entirety
as a feedstock is a key advantage inherent to gas fermentation
compared to sugar and cellulosic fermentation to produce lowcarbon fuels.

12. Unique features of gas-fermenting bacteria – acetogens

Acetogens are gas-fermenting bacteria with unique metabolic pathways, such as the Wood-
Ljungdahl Pathway (WLP). These bacteria can convert gases like CO and H2 into valuable
products like acetate and ethanol.
Module 7

Study materials (uploaded in Moodle):

13. Energy conservation in acetogens

Acetogens employ various energy conservation mechanisms, including the Wood-Ljungdahl

Pathway and Flavin-based electron bifurcation, to generate ATP and drive essential cellular
processes.

14. Importance of stoichiometry and mass balances in systems biology

Stoichiometry and mass balances are fundamental in systems biology for understanding the
flow of metabolites in cellular networks. They provide a quantitative basis for modeling and
analyzing cellular processes.

15. Concept and importance of chemostat for systems biology

The chemostat is a continuous culture system used in systems biology to maintain a steady-
state condition. It enables the study of microbial growth under controlled conditions,
facilitating the analysis of metabolic fluxes and system behavior

16. Basics of cultivation stoichiometry, including gas fermentation

Cultivation stoichiometry involves understanding the relationships between nutrient inputs

and microbial growth. In gas fermentation, stoichiometry is crucial for balancing gas
consumption and product formation, providing insights into the efficiency of the process.

17. Application of systems biology for gas fermentation

Systems biology is applied in gas fermentation to integrate various -omics data, build
predictive models, and gain insights into the metabolic pathways of gas-fermenting
microorganisms. This approach aids in optimizing conditions for improved product yields

18. The design-built-test-learn engineering concept

The design-built-test-learn (DBTL) engineering concept is an iterative approach in synthetic

biology and metabolic engineering. It involves designing a system, constructing it, testing its
performance, and learning from the results to improve the design in subsequent iterations.

19. Importance of systems biology in the design-built-test-learn engineering concept

Systems biology plays a crucial role in the DBTL engineering concept by providing a holistic
understanding of cellular processes. It guides the design of genetic constructs, helps in
predicting system behavior, and informs the iterative optimization process

20. Applications of systems biology in industrial biotechnology

Systems biology has numerous applications in industrial biotechnology, including strain

optimization, pathway engineering, and the development of bioprocesses. It contributes to
the efficient design and production of bio-based products in various industries.