Genetics Chapter on Homologous Recombination

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Questions and Answers

What is one of the main uses of homologous recombination in cells?

Transcription of RNA

Repair of chromosomal double-strand breaks (correct)

Replication of DNA

Introduction of new genetic information

Which genetic tool is commonly used for inducing double-strand breaks at specific loci?

TALENs

RNA interference

CRISPR/Cas9 (correct)

Zinc finger nucleases

What must be provided to the cell after inducing a double-strand break for homologous recombination to occur?

An RNA guide molecule

A linear double-stranded DNA fragment homologous to the break (correct)

A circular DNA fragment

A plasmid vector

In addition to repair, what is another important role of homologous recombination?

Generating cross-overs during meiosis Signup and view all the answers

Which scenario represents the use of advanced genetic tools for targeted integration?

Introducing a DNA fragment at a specific chromosomal location Signup and view all the answers

What is a potential evolutionary benefit of cross-overs during meiosis?

Creation of new genetic combinations Signup and view all the answers

What is one way researchers can 'cheat' during homologous recombination?

Providing a fragment homologous only at the ends Signup and view all the answers

What is an essential step after achieving chromosomal modifications in the lab?

Diagnostic PCR to verify integration Signup and view all the answers

What is the primary product formed when 1 mol of NADP+ is reduced per mol of glucose 6-phosphate?

NADPH Signup and view all the answers

What distinguishes an anabolic metabolic pathway from a catabolic one?

Anabolic pathways synthesize complex molecules. Signup and view all the answers

Which enzyme is specifically responsible for the conversion of glucose 6-phosphate in the indicated reaction?

G6PDH Signup and view all the answers

Which of the following is NOT a class of enzymes typically used in industrial applications?

Hydrolyses Signup and view all the answers

Which of the following best describes metabolomics?

The analysis of the complete set of metabolites in a biological system. Signup and view all the answers

What is a significant advantage of using extracellular enzymes over intracellular enzymes in industry?

They can be recovered from the surrounding medium easily. Signup and view all the answers

Biomass in biotechnology primarily refers to what?

Mass of living or organic material in an environment. Signup and view all the answers

What role do chiral centers play in biochemistry?

They create non-superimposable mirror images that affect enzyme interactions. Signup and view all the answers

Which enzyme type catalyzes electron transfer reactions?

Oxidoreductases Signup and view all the answers

What characteristic makes extracellular enzymes suitable for industrial applications?

Their high stability in harsh conditions. Signup and view all the answers

Which statement about enantiomers is accurate?

They are mirror images of each other and can display different biological activities. Signup and view all the answers

What is the primary function of hydrolases in industrial processes?

To break down large molecules using water. Signup and view all the answers

Biocatalysis relies on what type of molecules to perform chemical reactions?

Natural enzymes or whole cells. Signup and view all the answers

Which of the following statements about co-factors in enzymatic reactions is incorrect?

They are consumed in the reaction. Signup and view all the answers

Which of the following processes is a catabolic metabolic pathway?

Glycolysis. Signup and view all the answers

What is the significance of energy transfer in metabolism?

It maintains life by enabling the breakdown and synthesis of molecules. Signup and view all the answers

What is the primary difference between respiration and fermentation in terms of ATP generation?

Respiration involves electron transfer to oxygen for ATP generation. Signup and view all the answers

Which of the following statements accurately describes the Crabtree effect?

It describes fermentation occurring even when oxygen is available. Signup and view all the answers

Which pathway is primarily responsible for the conversion of glucose to pyruvate?

Emben-Meyerhof pathway Signup and view all the answers

What defines an amphibolic pathway in metabolism?

It can participate in both catabolic and anabolic processes. Signup and view all the answers

What is the role of NADH in fermentation?

It donates electrons to carbon intermediates. Signup and view all the answers

What was the outcome of the experiment with the swan-neck flask?

The broth remained clear and sterile. Signup and view all the answers

What conclusion was drawn from the swan-neck flask experiment?

Microorganisms originate from the environment. Signup and view all the answers

How did Eduard Buchner demonstrate that yeast enzymes are responsible for fermentation?

He isolated enzymes from the yeast extract. Signup and view all the answers

What does biotransformation specifically refer to?

Chemical modification of compounds by biological agents. Signup and view all the answers

Which statement accurately defines de novo biosynthesis?

It involves constructing molecules from simple materials. Signup and view all the answers

What is a primary use of cell-free enzymes in bioprocesses?

To accelerate biological reactions without whole cells. Signup and view all the answers

In the concept of whole-cell biotransformation, what role do the enzymes inside cells play?

They catalyze the reactions by themselves. Signup and view all the answers

What is fermentation according to the content provided?

A chemical conversion facilitated by specific compounds. Signup and view all the answers

What is a primary advantage of chromosomal modifications over plasmid-based systems in metabolic engineering?

Chromosomal integrations are more stable than plasmids. Signup and view all the answers

Why are chromosomal modifications considered to have a reduced metabolic burden compared to plasmid-based expression systems?

They do not require maintenance of additional genetic material. Signup and view all the answers

What advantage do chromosomal modifications offer in terms of gene loss during cell division?

They have a stable inheritance in daughter cells. Signup and view all the answers

Which of the following is a disadvantage of plasmid-based expression systems?

Plasmid size is limited due to instability. Signup and view all the answers

In the context of repairing DNA double-strand breaks, which process is essential besides non-homologous end-joining?

Homologous recombination Signup and view all the answers

Which statement best describes a feature of plasmid-based systems in metabolic engineering?

They allow manipulation of multiple copies of genes. Signup and view all the answers

What makes chromosomal modifications more suitable for industrial-scale applications?

They are less prone to variations in gene expression. Signup and view all the answers

In which situation are plasmid-based systems typically less favorable than chromosomal modifications?

When long-term stability of modifications is required. Signup and view all the answers

Study Notes

Industrial Biotechnology

Reflects the status quo of the current chemical industry in terms of sustainability, including raw materials, carbon sources, catalysts, energy consumption, and waste streams.
White biotechnology is the largest area of biotechnology, focusing on the use of biocatalysts for industrial-scale production and processing of products.
It aims to reduce the environmental impact of industrial processes and encourage the use of biodegradable polymers and renewable fuels instead of fossil resources.
Traditional chemical production methods often aren't suitable for renewable carbon sources.
Biotechnology offers diverse solutions for a sustainable chemical industry, including removing pollutants and preventing future pollution.
Biocatalyst-based processes are more efficient and environmentally friendly than conventional methods.
Biocatalysts tend to be self-replicating.

Fossil Carbon

Carbon stored in fossil fuels (coal, oil, and natural gas) formed from ancient plant and organism remains.
Fossil carbon represents carbon sequestered over geological time.

Renewable Carbon Sources

Renewable carbon sources are derived from sustainable and regenerative processes, unlike fossil carbon which is finite and non-renewable.
Examples include biomass (plant, tree and agricultural crops/organic waste) and biochar (produced via biomass pyrolysis).
Renewable carbon sources address environmental concerns, including reducing greenhouse gas emissions, promoting circular economy principles, and mitigating climate change.

Enzymes in Industrial Biotechnology

Enzymes act as catalysts in chemical reactions, either in an isolated form (cell-free) or within cells.
Industrial biotechnology commonly leverages microorganisms and their enzymes for various applications.

Yeast Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae is vital in industrial biotechnology as a cell factory.
Yeast's features (eukaryote, robustness, Crabtree-positive, facultative anaerobe, extensive metabolic engineering accessibility) contribute to its use in large-scale fermentations.

Biocatalysis

Biocatalysis is the use of natural catalysts like enzymes or whole cells to perform chemical reactions.
Biocatalysis is often more specific, efficient, and uses milder conditions than traditional chemical methods.
Examples of biocatalysis applications include synthesis of pharmaceuticals, production of biofuels, and environmental cleanup.

Biocatalysts

Biocatalysts encompass any biological molecule or system that accelerates chemical reactions, potentially including isolated enzymes, extracellular enzymes, and whole cells (e.g., microorganisms)

Bioprocesses Part A

Biotransformation
- Modifies compounds using biological agents (enzymes/microorganisms), typically to create more valuable/functional products.
De Novo Biosynthesis
- Creating complex organic molecules from simple precursors using metabolic pathways.
Fermentation
- Uses microorganisms to convert organic compounds (like sugars) into simpler substances in the absence of oxygen, generating energy.
Whole-Cell Biotransformation
- Utilizes intact living cells as biocatalysts with metabolic pathways and enzymes for transformations.

Bioprocesses Part B

Bioreactors offer advantages over smaller vessels like microtiter plates and shake flasks for large-scale bioprocesses (controllable conditions for oxygen, mixing, pH, continuous feeding).
Different cultivation modes (batch, fed-batch, continuous)
Batch mode involves a single medium addition; no feeding.
Fed-batch mode involves adding medium incrementally when substrate levels are low.
Continuous mode involves constant feeding and product removal to maintain a steady-state.

Product Titer, Yield and Production Rate

Titer: Concentration of product
Yield: Ratio of product formed to substrate consumed (g product/g substrate).
Volumetric Productivity: Conversion rate per unit volume
Specific Productivity: Conversion rate per unit biomass

Downstream Processing

Downstream processing (after bioconversion) handles separation and purification of products, which is crucial in evaluating total production costs.
Extracellular products often have simpler processing compared to intracellular ones, which require cell disruption.

Enzymes and Cofactors Part A

Enzymes reduce activation energies for chemical reactions without altering equilibrium constants. Enzymes stabilize transition states.
Enzymes are classified by the type of reaction catalyzed. (Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, Ligases).
EC numbers provide a universal coding system for identifying and classifying enzymes.
BRENDA is a useful database for obtaining information about enzymes
Cofactors (coenzymes and prosthetic groups) often required for enzyme function, including vitamins and metal ions.

Enzymes and Cofactors Part B

Units (U) are the standard units of enzyme activity.
Optical tests (like coupled enzyme assays) use the characteristics of cofactors (like NAD(P)+/NAD(P)H) absorbance changes to measure enzyme activity quantities.

Large-Scale Industrial Biotransformations Using Industrial Enzymes

Extracellular enzymes are more often favored over intracellular ones due to easier recovery, reduced costs for production, and higher stability in harsh conditions, as well as continuous production strategies.

Examples of Industrial Enzyme Applications

Examples include glucose isomerase for syrups, pectinase for fruit processing, lactase for lactose-free products, and phytase for animal feed.

Cofactor Recycling Systems

Cofactor recycling systems for biotransformations enhance efficiency and reduce costs by enabling the continuous supply of essential cofactors (e.g., NAD(P)H and ATP).
Secondary enzymes regenerate the initial cofactor using cheap substrates. (e.g., lactate dehydrogenase regenerates NADH)
Cofactor recycling is crucial for both fermentation and respiration.

Non-Fossil Substrates

Non-fossil substrates include sugars (e.g., sucrose), starch-based materials using enzymes (a-amylase, glucoamylase, pullulanase), and lignocelluloses (cellulose, hemicellulose, lignin).
Starch is easier to convert into glucose than lignocellulose due to structural differences in the carbohydrate molecule.

Metabolic Engineering

Metabolic engineering involves the deliberate modification of a microbe’s metabolism to enhance production pathways, improve efficiency, or adapt under specific conditions, involving use of recombinant technology, and is used in industrial contexts.
Methods to manipulate enzyme activities include overexpression, activity reduction, and introducing heterologous enzymes.

Chromosomal Engineering

A strategy used to modify microbial genetic material for targeted modifications (deletions, insertions, replacements) requiring homologous chromosomes, including use of tools (like CRISPR/Cas9) used to induce targeted double strand breaks.
Chromosomal integration offers advantages for production, stability (reducing the need of continuous selection), and scalability, which is vital for industrial applications.

Central Metabolic Pathways: Respiratory Chain and Oxidative Phosphorylation

Electrons from energy-rich compounds enter the respiratory chain.
Electron flow drives proton transport (for creating the proton motive force/electrochemical gradients/energy currency in cells) ultimately involving electron donation from organic molecules to oxygen in aerobic respiration).
The proton motive force is used to synthesize ATP (via ATPase/ATP synthase).

Cellular Metabolism and Fermentation

Fermentation is a metabolic process that does not require oxygen but utilizes an organic compound as a terminal electron acceptor.
This process generates energy through substrate-level phosphorylation by directly transferring phosphate from a phosphorylated intermediate to ADP.
Both fermentation and respiration pathways work in concert, depending on available substrates and environmental conditions.

Pentose Phosphate Pathway (PPP)

The pentose phosphate pathway (PPP) is an anabolic/catabolic pathway, generating intermediates, reducing power (NADPH), and ATP.
The pathway is two-phased (oxidative and non-oxidative).
Provides precursors for nucleotide and amino acid biosynthesis.

Growth of Microorganisms on Different Carbon Sources

Cells can adapt to different carbon sources, such as C1 compounds (e.g., methanol).
Specialized enzymes are used for handling different carbon substrates, which are then often funneled into central metabolic pathways (e.g., TCA cycle) for energy production and synthesis of biomass compounds.
Utilization of diverse carbon sources is particularly crucial for the industrial use of microorganisms for chemical production.

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Test your knowledge on the mechanisms and applications of homologous recombination in cells. This quiz covers topics such as double-strand breaks, genetic tools used in research, and the roles of cross-overs in evolution. Perfect for students studying genetics or molecular biology.