Advanced Biotechnology

In the following case: "A microbial host needs to be engineered for 100 g/L production of a target chemical, but a concentration of 50 g/L of the same chemical has a toxic effect on the cell". How to make this microbe a better production host and increase its tolerance to the target chemical by means of (a) combinatorial engineering and (b) process engineering

(a) combinatorial engineering - By using the adaptive laboratory evolution, one could isolate strains with tolerance to the taxic effects at 100g/L. These strains can then be analysed to further reveal molecular mechanisms of the resistance, which can be added to the host strains through rational engineering. (b) process engineering - The different molecular mechanisms must be studied to find resistance mechanisms that could work. After finding this mechanism the host strains are engineered to tolerate the toxicity

What happens when the Cas9 protein makes a double stranded cut in the DNA in a eukaryotic cell?

- After the cut, the cell will try to repair the damaged DNA. If there is a complementary repair template is available it will be inserted in the cut site, but it there isn't the cell will use the non-homologous End-joining pathway. Where it ligates the to ends to each other.

Briefly explain the four RRI dimensions (what are they and what do they mean?).

- Anticipation ( to look forward). It has been proven difficult to develop substantive diagnoses and correspondingly good prospects. Anticipation is about how actors in science and innovation should map the plausible effects of their innovations, intended as well as unintended ones, and develop socially robust strategies to prevent harmful and undesirable outcomes. - Reflexibility, (think through). A greater degree of reflexivity is vital in order to provide directionality in research and innovation processes. Scientists are invited to evaluate their own moral, political and social assumptions because these can have an influence on the choice for research problems, methods and innovation design. Expertise and capacity is important to identify and discuss prior knowledge in the form of fundamentals, assumptions and framework of understanding, smaller uncertainties and areas of ignorance. - Inclusion, (invite along). Encourages research and innovation actors to get in touch with potential future users and other concerned actors. They can provide valuable insights into contexts of application as well as their opinions on desirable research approaches. Enables society to speak back to science, it has increased and been directed toward the research environment itself. This is important for opening up research and innovation processes, recognizing the limits of one's own knowledge and competence, and the ability to ask for help in dealing with the potentially landscape-changing effects of these processes. - Responsiveness, (work together). The first three dimensions are intended to provide continual input and substance to new governance practices. Responsiveness is about the development of horizontal or distributed governance schemes that encourage collaboration with partners that may be affected by a research and innovation process. Responsiveness urges scientists to change research and innovation trajectories if the feedback from stakeholders or public opinion show that present goals and planned actions are contrary to social needs, or are ethically unacceptable. This takes place via broad-based involvment, and can include policy actors, research counsils, trade and industry, interest organisations and society. The underlying question all researchers and innovators should ask themselves is: what kind of a future do we want to create through research and innovation?

Describe the general selection criteria that are typically applied and explain how they work.

- By providing the electron acceptors and donors needed for the desired conversions - By selecting for populations that are able to grow attached (flocs/biofilms/granules at low HRT) - Other selective forces: interactions between microbial populations, predation, viruses, un-controlled changes of environments, etc. - Therefore, the exact microbiome composition will vary over time and between systems of the same process

How does cells remodel their own microenvironment and how can we use this knowledge in our tissue engineering strategies?

- Cells are consistently rebuilding the ECM by synthesis, degradation, reassembly, and chemical modifications. - Degradation of the EMC occurs through different component, such as proteases that degrade the proteins in the ECM. Another effective strategy to remodel the ECM is to modulate the levels and organization of ECM components assembled within the protein network, though ECM modifieng enzymes. - This can be used to modify the cells to be able to recreate their microenvironment. By ex. Inducing the cell with signals that promote ECM synthesis.

What is the extracellular matrix (ECM)? Explain how natural ECM can be used as in inspiration to create synthetic mimics for tissue engineering.

- ECM: A large network of proteins and other molecules that surround, support, and give structure to cells and tissues in the body. o Cell anchorage o Channel for signaling molecules o Growth factor receptors and reservoir for growth factors o Tissue remodelling and homeostasis o Facilitates cell migration

Given the advantage of the available tools and massive background knowledge on the metabolism of model organisms, e.g. Corynebaterium glutamicum and Escherichia coli, explain the reasoning for exploring alternative and rather unestablished production hosts in metabolic engineering.

- Exploring established production hosts can lead to finding of hosts with ability to grow in specific environments (such a lower pH or high temperature), use variety of feedstocks or naturally produce and tolerate large amounts of a desired product. This would lead to improvement in production or more cost-effective production.

Industrial biotechnology typically scale up recombinant protein production by growing cells in so-called fermentors. Describe advantages and challenges by cultivating recombinant E. coli in fermentors and describe why it is particularly useful to use inducible expression systems under such conditions

- Fermentor = bioreactor for controlled cultivations of bacteria to high-cell-densities (HCD) Cultivating recombinant E. coli in fermenters (bioreactors): ● Challenges: - HCD confers extra stress and burden to cells ○ strong selection against recombinant cells ○ plasmid loss is a huge problem - limitation and/or inhibition of substrates (som substrates are added later) - limited capacity for oksygen - formation of metabolic byproducts ● Advantages: - low costs - fast growth rate (high efficiency) - ease of control (temperature, medium, aeration, pH) - lower space requirements - high acetic acid concentrations - simplified purity Why it is particularly useful to use inducible expression systems in such reactors: - Can control the amount of recombinant bacteria produced, by adding an inducer. Expression turned off when the inducer is not added.

A heterologous gene originating from a yeast is to be expressed recombinant in the bacterium Escherichia coli. a. Describe genetic modifications needed to achieve this

- Genetic modification needed to achieve this: In this case the recombinant expression I of eukaryotic genes (plant) in E.coli. The challenges is the intro/exon and spicing, since bacteria lack the enzymes needed for mRNA spicing. One could use produce cDNA of the mature mRNA using reverse transcription. Eukaryotic DNA strand àtranscription àRNAà Splicing à mRNA à reverse transcriptase à cDNA (inserted into a plasmid)

What are iPSC? What are sources for iPSC? What can they differentiate into? What are benefits compared to ESC?

- IPSC (induced Pluripotent Stem cells. Specialized (adult) cells with pluripotency induced by genetic engineering or soluble factors. - Pluripotent cells can give rise to virtually all cell types in the body. Common sources of the IPSC are somatic cells (blood and skin cells that have been reverse engineered). - IPSC vs ESC: - The ethical question that arise when one is using ESC are one of the downfalls of that. - IPSC from derived from the patients cell would have perfect histocompatibility.

You manage a) but you observe low expression level of your heterologous gene product; describe three different strategies you can test to increase expression level

- Improvement of recombinant expression relies on the modulation of circumvention of may issues, such as mRNA stability, codon bias and inclusion body formation. Several strategies have been made for efficient production of proteins in E. coli, namely, the use of different mutated host strains, co-production of chaperones and foldases, lowering cultivation temperatures, and addition of a fusion partner. Stability of the recombinant mRNA: The average halflife of mRNA in E. coli at 37 ◦C ranges from seconds to maximally 20 min and the expression rate depends directly on the inherent mRNA stability. Solution: - the stability can be modulated by genetic engineering: - use efficient rbs - ribosome protection of mRNA degradation - host strains deficient in RNAse genes (mangler nok RNAse genes?) Codon usage - codons rare in E. coli are often abundant (rikelig) in heterologous genes from f.ex. eukaryotes. - expression of genes containing rare codons: → low availability of complementary tRNAs, so rare codons slow down translation rate. The rare codons can lead to translation errors as a result of ribosomal stalling at positions requiring incorporation of amino acids coupled to minor codon tRNAs. Solution: Redesign coding region to avoid rare codons without changing the gene product by gene synthesis. - site-directed mutagenesis of the target sequence for the generation of codons reflecting the tRNA pool in the host system (E. coli). This approach is beneficial for increasing expression levels and for alleviation of mistranslation. However, a set of codon-optimized genes was recently shown to suffer from lacking mRNA transcription and stability in a recombinant expression system... It's also a highly effective method, but it may be too time-consuming in high throughput biotechnology. OR: host introduced genes encoding rare tRNAs - less time consuming method - co-transformation of the host with a plasmid harbouring a gene encoding the tRNA cognate to the problematic codons - By increasing the copy number of the limiting tRNAspecies, E. coli can be controlled to match the codon usage frequency in heterologous genes. Prevention of inclusion body (IB) formation Protein activity depends on precise protein folding. Stress situations such as heat shock, impairs (svekker) folding in vivo and folding intermediates tend then to associate into amorph protein granules termed inclusion bodies. Inclusion bodies are complex aggregates and are often seen as a result of stress, when recombinant protein is expressed at high rates. These inclusion bodies are non-functional and non-soluble. Cytoplasmic expression often results in formation of IBs. Solutions: - co-expression of chaperones (assists folding) - expression at low temperature (slows down translation rate) - export to periplasm (if recombinant protein has di-sulphide bonds)

Describe the 4 fundamental concepts in constraint-based reconstruction and analysis methods

- Network constrains: Imposition of physiochemical constrains that limit the computable phenotypes, these concerns: mass balance, charge balance and thermodynamics - Objective functions: The identification and mathematical description of evolutionary selective pressure. There are several pathways to a product and due to evolution, the cell will have developed a regulation of these (on/off mechanisms). If we can find these, we can modulate which pathway the cell will use under specific conditions that we set. - Association of reactions with the genome: A genome-scale perspective of the cell metabolism that accounts for all the metabolic gene products in a cell. If we have the whole genome available and every gene functions found, we can modulate a pathway from the genome level to the phenotypic level.

Which aerobic microbial process is commonly applied to convert toxic ammonia to nitrate in the biofilters of recirculating aquaculture systems (RAS)? How is this microbial process selected for?

- Nitrifying bacteria utilize ammonia and nitrite as energy source. - Selection: - Short hydraulic retention time - Provide surfaces for biofilm growth in the biofilter - Supply of O2 (e-acceptor) through aeration - Presence of ammonia (produced by the fish) - Low levels of readily degradable organic compounds

Do the CRISPR/Cas enzymes only target DNA or can they also target other biomolecules? If so, what can they target and what are the potential biotechnological applications?

- RNA?

Your gene product is host-toxic; describe two different strategies that can be done to overcome this problem

- Solution generally about stress responses (including toxic gene product) - Tightly regulated promoter - Grow recombinant cells to high densities under low/no background expression (can use promotors that rely on positive control à lower background expression. ) - Turn on recombinant production by induction.

A well-functioning human gut microbiome is highly important for human health, whereas dysbiosis in the gut microbiome is associated with diseases. b. Discuss how this might be obtained, and reasons why this might be difficult.

- Some factors as genetics and age, cannot be controlled and hence hard to change microbiome composition.

Explain the differences and advantages between flux balance analysis (FBA) in comparison to metabolic flux analysis (MFA). Explain the outcomes on each technique and in which situations each of them is applicable.

- The key difference between metabolic flux analysis and flux balance analysis is that metabolic flux analysis is an imaging fluxomics technique for analyzing production and consumption rates of metabolites in a biological system, while flux balance analysis is a mathematical fluxomics technique for analyzing the flow of metabolites through a metabolic network. Fluxomics refers to various approaches used to determine the rates of metabolic reactions within a biological system. - MFA: This technique allows for the quantification of metabolites at an intracellular level. Therefore, this technique elucidates the central metabolism of the cell. In metabolic flux analysis, the 13C fluxomics based method is commonly used for flux analysis. - FBA: it is possible to predict the growth rate of an organism or the production rate of a biotechnologically important metabolite through FBA.

The activated sludge is a biological wastewater treatment process that has been used to treat municipal wastewater around the world for a century. a. Explain why biomass growth in flocs is essential in this process

- floc formation is essential for the sedimentation to occur. If there are no flocs the treated water would be dragged down together with the waste and the system would not work.

Can you suggest a strategy to select for denitrification in RAS? Answer briefly.

- reducing O2

"Science consists of choices". Briefly discuss this statement and give at least two examples of choices taken in science.

- science consists of choices: methods, theories, literature and models - choices have social, economic, political, cultural, ecological implications - RRI, thinking about how these choices change and shape research. .......

The activated sludge is a biological wastewater treatment process that has been used to treat municipal wastewater around the world for a century. b. Describe how floc formation is promoted in activated sludge systems

Adjusting sludge age and starvation leads to more floc formation. Reduction of oxygen level leads to lower degradation of organic compounds and hence keeps the biomass in the system.

5) A) Name three synthetic biology approaches within microbial consortia and explain in detail of them? B) Name three synthetic biology tools to engineer and control microbial communities and explain in detail of them? C) What are the advantages of using microbial consortia?

A) Name three synthetic biology approaches within microbial consortia and explain in detail of them? - Population control: Cell populations in coculture can be engineered to express a different QS system that self-induces lysis upon reaching a tuneable population threshold leading to oscillations in the population level of each strain. - Distribution of tasks / Pathway separation: Reduction of the metabolic load, or burden, placed on any given organism. - Spatial programming: Intercellular signalling mechanisms between cells and exogenous inputs to control gene expression can be used to form distinct spatial patterns and morphologies in synthetic microbial consortia by activating the expression of adhesins, ligand receptors, or other polymers. B) Name three synthetic biology tools to engineer and control microbial communities and explain in detail of them? - Intercellular signaling: Quorum sensing (QS) systems can be used to coordinate signaling between organisms. In the simplest case, a signaling molecule is produced LuxI by a 'sender' cell, diffuses through the cell membrane, binds to its corresponding receptor (luxR), and activates transcription of the Plux promoter in the 'receiver' cell. - Exogenous molecules: Gene expression in strains within a synthetic consortium can be independently regulated via exogenous addition of inducer molecules Induction of a QS system via IPTG in one strain can also be used to trigger protective mechanisms, such as expression of an ampicillin resistance gene (AmpR), in a second strain expressing the corresponding QS receptor. This enables mutual survival of a consortium in the presence of an antibiotic, such as ampicillin. Unannotated promoters are constitutively expressed. - Syntrophic interactions: The resources produced by one organism are used by the other and vice versa. Additionally, synthetic consortia can be assembled by deleting essential genes, typically amino acid biosynthesis genes, in each member. Survival of each member thus becomes dependent upon resource sharing from other strains in the culture. This approach enabled a 14-member Escherichia coli consortium to be assembled. Each letter indicates an amino acid that has been deleted in the organism and straight lines indicate resource sharing between the strains. C) What are the advantages of using microbial consortia? -

A) What is a microbial consortium? B) What are the advantages of using microbial consortia? C) Explain how a 3-organism consortium could be established via syntrophic interactions. D) What does it mean distribution of tasks in the context of microbial consortia?

A) What is a microbial consortium? - A microbial consortium is two or more bacteria or microbes living symbiotically. B) What are the advantages of using microbial consortia? - Division of labour between the different groups. - Spatial organization. - Robust against environmental changes and contamination. C) Explain how a 3-organism consortium could be established via syntrophic interactions. - The resources produced by one organism are used by the other and vice versa. Additionally, synthetic consortia can be assembled by deleting essential genes, typically amino acid biosynthesis genes, in each member. Survival of each member thus becomes dependent upon resource sharing from other strains in the culture. D) What does it mean distribution of tasks in the context of microbial consortia? - Reduction of the metabolic load, or burden, placed on any given organism. This means that one group is responsible for production and/or degradation of set of metabolites, meanwhile the other groups are responsible for other sets.

A) What kind of functional element is depicted in the picture? B) How does it regulate genetic expression? C) which kind of logic gate represents and why? D) What are the differences between the logic gates NAND and NOR?

A) What kind of functional element is depicted in the picture? - Riboswitch B) How does it regulate genetic expression? - When the ligand binds the mRNA changes confirmation, so that the Ribosome binding site is no longer exposed. This means that the ribosome cannot bind to the mRNA and the translation is inhibited. C) which kind of logic gate represents and why? - This is a NO gate, because the presence of the ligand leads to inhibition of translation. And in the absence of the ligand translation would occur. D) What are the differences between the logic gates NAND and NOR? - NAND: In this logic gate the presence of both inducers inhibits transcription. The inducers inactivate the repressor and lead to the activation the NO-gate. The NO-gate inhibits transcription. - NOR: In this gate both activators have to be absent for transcription to happen. The NO-gate is activated by the OR-gate and the absence of the inducers would deactivate the OR-gate and in turn the NO-gate.

4) A) Why to create a minimal cell? B) What advantages genome reduced organisms have? C) What properties a target organism should have to create synthetic cells by top-down approach?

A) Why to create a minimal cell? - To define a minimal set of genetic functions essential for life under ideal laboratory conditions. • To discover the set of genes of currently unknown function that are essential and to determine their functions. • To have a simple system for whole cell modelling. • To modularize the genes for each process in the cell (translation, replication, energy production, etc.) and to design a cell from those modules. • To build more complex cells by adding new functional modules. B) What advantages genome reduced organisms have? - Cells with reduced genomes grow faster, utilize metabolic resources better, easier to control, have a more stable genome and less bi-product production. C) What properties a target organism should have to create synthetic cells by top-down approach? - The target organism should have a small genome that has been sequenced. Should also be a well characterized and easy to grow in a lab. There should also be genome editing tools that can be used on the target organism.

Choose one functional element that can regulate gene expression: A) explain the selected functional element. B) Which kind of logic gate represents and why? C) What are the differences between the logic gates AND and OR? D) What kind of expression pattern of the reporter protein GFP would you expect from the following genetic circuit scheme and why?

A) explain the selected functional element. - Riboswitches are regulatory segments of a messenger RNA molecules that bind small molecules, resulting in a change in production of the proteins encoded by the mRNA. They regulate gene expression through translation inhibition. B) Which kind of logic gate represents and why? - NOT gate. The presence of the small molecule prevents translation, hence riboswitches are repressor complexes. C) What are the differences between the logic gates AND and OR? - In the AND gate both inducers are required to expose the promoter (activate), this is achieved by inactivating the repressor. - In the OR gate only one on the inducer is needed to inactivate the repressor. The presence of both inducers would lead to further repression. D) What kind of expression pattern of the reporter protein GFP would you expect from the following genetic circuit scheme and why? - The GFR protein would have an osculating expression pattern. The repressors are arranged in a ring form which leads to an osculating expression because of the circular nature or the repression. The expression of R2 leads to inhibition of R3 and expression of GFR, but the absence of R3 leads to the expression of R1 which inhibits R2, and lack of R2 leads to expression of R3. R3 inhibits the GFP and R1, which leads to the expression of R2.

The activated sludge is a biological wastewater treatment process that has been used to treat municipal wastewater around the world for a century. Which type of microbial conversions are dominating this process

Anoxic and aerobic respiration dominate this process

Explain why so many different microbial processes take place simultaneously in the granules.

Because of the different environmental conditions (o2 availability) and different prosses can take place at once without disturbing each other.

What material properties are important to consider when selecting a material for tissue engineering applications?

Biocompatibility - Mininal adverse reactions in the host Biodegradable - Degradation rate vs growth rate Surface properties - Promote cell adhesion - cell infiltration and tissue integration Mechanical performance - strength to withstand physiological forces Clinical feasibility - Ability to be applied in surgical setting - Cost

Inactivation of genes has been an important tool to study the functions of genes in many model organisms. What is the advantage of the CRISPR/Cas9 technology compared to the previous knockout methods?

CRISPER/Cas9 is cheaper and easier to handle than the previous methods. The Cas9 nuclease uses a guide RNA, which can be modified to target specific locations in the genome. In contrast to zinc fingers and TALENs which rely on protein-DNA interactions for targeting

If denitrification took place in the biofilter, nitrogen would be removed from the water as dinitrogen gas. This would improve the water quality. Would you expect denitrification to take place in the RAS biofilter? Justify your answer.

Denitrifying bacteria are usually anaerobic, and the denitrification can take place in the biofilters only if the environment is anaerobic, which is not the case in the RAS system.

A well-functioning human gut microbiome is highly important for human health, whereas dysbiosis in the gut microbiome is associated with diseases. a. Is it possible to steer the human gut microbiome towards a healthy microbiome composition?

Diet and lifestyle are two important factors that can influence the gut microbiome composition and altered lifestyle and diet can lead to a healthier gut microbiome composition. - Fecal transplantation is shown to revert dysbiosis after antibiotic treatment.

What are the European Commission six RRI policy keys? Choose two of the keys and give a more detailed explanation of these.

Ethics. This focuses on research integrity. the prevention of unacceptable research and research practices . Research integrity refers to the appropriate conduct of research to prevent misconduct or negligence. This includes both the research that is carried out, but also the researchers behavior. It also focuses on science and society, and the ethical acceptability of scientific and technological developments. Gender equality. This is about promoting gender balanced teams, ensuring gender balance in decision-making processes and always considering the gender dimension in research and innovation to improve the quality and social relevance of the results. Society is 50% women, but especially at a higher level, the majority within research science is male. This could without intention affect the research questions asked and the models built. Governance: A focus on governance arrangements that allow for acceptable and desirable futures have to be robust and adaptable to the unpredictable development of R&I (de facto governance); governance should be familiar enough to align with existing practices in R&I; governance should share responsibility and accountability between all actors; and provide governance instruments to actually foster this shared responsibility. 4. Open Access: Should address issues of accessibility to and ownership of scientific information. Free and earlier access to scientific work may improve the quality of scientific research and facilitate fast innovation, constructive collaboration among peers and productive dialogue with civil society. 5. Public Engagement: There should be a fostering of R&I processes that are collaborative and multi actor: all societal actors work together during the whole process in order to align its outcome to the values, needs and expectations of society. 6. Science Education: A focus on science education which enhances the current educational process to better equip citizens with the necessary knowledge and skill so they can participate in R&I debates; and increasing the number of researchers (a promotion of scientific vocations).

Fusion technology is a technique with many different applications; describe how this technique can be used to solve problems related: a. Protein solubility, b. Protein purification and c. Protein detection

Fusion protein technology is genetic engineering; short peptides ("tags") or entire proteins in-frame with recombinant protein are fused. - Recombinant fusion proteins are proteins made by recombinant DNA technology that involves joining of two or more genes from different sources, transcribed and translated as a single polypeptide. Fusion proteins usually include a partner/ a "tag" linked to the passenger or target protein by a recognition site for a specific protease. Most fusion partners are exploited for specific affinity purification strategies. Almost all recombinant proteins are prepared using fusion domains, also known as "tags" (protein tags). Protein tags refers to the sub-domain/the peptide sequence of a fusion protein. The tags are used as... Common affinity tags are the polyhistidine tag (His-tag), which is compatible with immobilized metal affinity chromatography (IMAC) and the glutathione S-transferase (GST) tag for purification on glutathione-based resins. Several other affinity tags exist and have been extensively reviewed a. Protein solubility Recombinant proteins are produced with poor solubility, so fusion tags are used to improve their solubility. A solubility fusion tag can be incorporated into the expression vector. Although many proteins are highly soluble, they are not all effective as solubility enhancers. E. coli MBP is an effective solubility enhancer. Solubility enhancement is a common trait of maltodextrin-binding proteins (MBPs) from a number of organisms and some of them are even more effective than E. coli MBP. A precise mechanism for the solubility enhancement of MBP has not been found. However, MBP might act as a chaperone by interactions through a solvent exposed "hot spot" on its surface, which stabilizes the otherwise insoluble passenger protein. The outcome of fusion to a solubility partner is protein specific and is not a universal method for the prevention of inclusion-body formation. b. Protein purification Fusion tags are used as affinity tags for protein purification. The recombinant protein to be produced is attached to a fusion tag (an affinity tag), ex. histidine. A chromatography step should first be conducted. The tagged protein can be easily and conveniently purified by affinity chromatography. Affinity chromatography: The sample is first bound to the ligand using favorable conditions for that binding. Then, the unbound material is washed out of the column and the elution of pure protein is achieved using a competitive ligand or by changing the pH, ionic strength, or polarity. This purification strategy can profit from the use of recombinant DNA technology as the affinity tag can be fused to the protein of interest during cloning and it is further presented in the next section. c. Protein detection To enable visualization of the location of proteins in a cell, tissue, or organism. Can use fluorescent tags or epitopes. Immunofluorescence microscopy is the most common technique for visualization.

What is the disadvantage of utilizing feedstocks based on glucose in biotechnological processes? Explain what can be done to decrease the use of glucose as feedstock in industrial biotechnology.

Glucose is the main energy source for both humans and animals, and high usage of glucose, ex. production of biofuel ethanol is going to have to compete with the human and animal nutrition. This leads to low availability of glucose. - The biotecknological production hosts E. coli, C. glutamicum, pseudomonads, bacilli and Baker's yeast used in these large-scale processes have been engineered for efficient utilization of alternative carbon sources. This reduces the glucose dependence.

mass and charge conservation

Mass conservation further limits the possible reaction products and their stoichiometry.

What is mechanosensing? Explain how mechanosensing can be used as a guide when engineering e.g. bone.

Mechanosensing describes the ability of a cell to sense mechanical cues of its microenvironment, including not only all components force, stress and strain, but also substrate rigidity, topology and adhesiveness. - Cells will sense mechanical features in their environment which leads to rapid motility and signal responses. This leads to the cell pulling on the environment and hence modifying the extracellular matrix and generate new signals, ex. Signal originating from unfolding of fibronectin. This leads to intracellular signalling pathways that alter the gene expression and lead to changes in cell shape, because of overtime cellular forces and cellularly generated matrices.

Reason why optimization of biomass production is a reasonable objective when modeling microbes

Modelling is a way to represent a "possible" reality. In a way, simulations may be run with mathematical models to visualise possible outputs from different inputs. This can be used as a way to plan how microbes can be optimised for biomass production. An example is a model which describes an evolutionary pressure for microbial growth and description of the metabolic demands to make the basic metabolite building blocks.

How to engineer the bacterium Corynebcaterium glutamicum in order to obtain increased lysine yield on glucose and how the metabolic flux of the newly engineered bacterial strain can be determined?

Overexpression of pyruvate carboxylase and aspartate kinase would increase the flux both into and out of the TCA cycle. This balances the intermediate concentration as it increases the lysine production and maintains the growth rate of the wild type strain. - The flux can be determined by measuring the lysine produced as well as the intermediates concentration.

Describe how codon usage can negatively affect recombinant gene expression and describe two different approaches that can be used to fix this

Rare codons that are not expressed in E.coli, mean that there would be lower amount or no matching tRNAs would lead to lower expression. - solutions: - redesign the codons so that there are no rare codon - Use a bacterial host with introduced rare tRNA genes.

substrate and enzyme availability

Substrates must be available in the microenvironment or produced from other reaction, and enzymes must be available. Absence of these would put limitation on the metabolic reaction as there would be no metabolic reaction without the enzymes or substrates.

Describe briefly the most relevant microbial processes taking place in aerobic granules in treatment of wastewater (the Nereda process).

The granules are divided into aerobic, anoxic and anaerobic zones. Aerobic zone: - Aerobic respiration (org. C → CO2) - Nitrification (aerobic respiration of ammonia; NH4 + → NO3) Anoxic zone: - Denitrification (anaerobic respiration; org. C + NO3 → CO2 + N2) Anaerobic zone: - Phosphorus accumulation by PAOs (need anaerobic and aerobic zones)

What is the role of CRISPR/Cas9 guideRNAs and how do they operate? Are there any requirements which needs to be considered for them to be functional?

The guideRNA is placed in the CAS9 protein is the part that binds to the target sequence in the DNA. The guideRNA contains about 20 nucleotides long targeting sequence, and this targeting sequence binds to the target in the DNA and allows the CAS9 to cut correct. - The gRNA should be around 100 nucleotides long and the targeting sequence should be 17-24 nucleotides. The targeting sequence should also have GC between 40-80% as higher GC content gives more stable bindings. - The non-complementary strand needs to contain a PAM site that follows the DNA target region. The PAM site is important for the cas protein to be able to cut the DNA.

Explain how to improve production of a target chemical by means of combinatorial methods of metabolic engineering?

The host used for production of target chemical is first exposed to mutations via UV-light or toxic reagents. This leads to many mutations and strains that have adapted a better production of the target chemical are further studied to understand what mutations led to a better production.

CRISPR is an abbreviation for "Clustered Regularly Interspaced Short Palindromic Repeats". What is the function of these repeats and how were they used to find the genes and proteins now commonly used for gene editing?

The spacers regions between the repeats are homologous to sequences bacteriophages, prophages and plasmids. These sequences work as an RNA guided defence system. The repeats together with the CAS9 protein work as an adaptive immune system.

A large continuous industrial flow of wastewater contains a slowly degradable contaminant, called ContA. This is the only organic compound in the wastewater flow. ContA is toxic and needs to be converted in a treatment process. A research group developed (through genetic engineering) a Pseudomonas strain that was able to degrade ContA at high rates, and this could potentially be used to treat the wastewater. Alternatively, a ContAdegrading microbiome might be possible to obtain through selection exerted by serving ContA as the only available electron donor. a. Which of these approaches would you chose for designing a treatment process for the wastewater? Justify your answer.

The microbiome system is preferred. The genetic engineered system is high-maintenance and expensive. Wastewater treatment is an open system and invasion of wild populations would quickly overturn the designer community with a natural microbiome, leading to unpredictable outcomes.

What is the objective of tissue engineering? What three components are usually seen as a part of tissue engineering strategies? What are the roles of these different components?

The objective of tissue engineering is to (re)generate new tissue. Regenerating a patient's own tissue and organs offers complete biocompatibility and biofunctionality and removes possibility of a severe immune response. Tissue engineering replaces structures in the human body, such as bones and tendons, or metabolism as found in the liver or pancreas. The alternatives to tissue engineering are organ transplantations or artificial organs. These have shortcomings like immune response as a result of rejection and sub-optimal biocompatibility and function. - The three components are: - Cells: Cells (stem cells) proliferate into more stem cells and differentiate into specialized cells and build tissue and organs. Restore tissue function - Scaffolds: Large tissues and organs have a distinct three-dimensional form, and hence regenerated cells need a form to support their formation. This support is called a scaffold and has majority similar functions as the natural extracellular matrix. It assists in proliferation, differentiation and biosynthesis of cells, and it prevents invasion of disturbing cells. The scaffold also gives mechanical support. - Bioactive factors/growth factors: Growth factors are proteins that induce tissue regeneration. They play a key role in proliferation and differentiation of cells.

3) Plasmids are commonly used as expression vectors: a. Describe similarities and differences between the pET and pBAD plasmid vectors for induced recombinant expression in Escherichia coli.

The pET expression system: Uses the hybrid T7/lac promoter on the plasmid. - T7 promoter from the bacteriophage T7 and lac promoter from E. coli. E. coli host with T7 RNA polymerase gene on chromosome under control of IPTG inducible lacUV5 promoter. Recombinant gene under control of T7/lac promoter on pET plasmid. Lacl gene is present on pET plasmid; encodes Lacl regulator (repressor). Lacl can bind to the promoter and repress T7/lac promoter and lacUV5 promoter. Inducer IPTG binds to Lacl regulator (repressor) → release it from lacUV5 and lac operator → RNA polymerase expression on→ T7 gene is transcribed → T7 RNA polymerase expression on → transcription from T7 promoter → recombinant expression turned ON → target gene transcribed. What's happening: We need to express the target gene inside E. coli, but to do this we also need to express the T7 gene, because the bacteriophage T7 gene will express the T7 RNA polymerase, which will be required for the expression of our target gene. IPTG inducer must be added (to take out the lac repressor) → when the lac promoter is free (not blocked) → T7 gene is transcribed in E. coli → T7 RNA polymerase is expressed→ transcription of target gene Lac repressors block the transcription of the target gene. Only when we want to produce the target gene IPTG inducer is added. The pBAD expression system: Expression plasmid using araBAD promoter, are designed for tight control of background expression and L-arabinose dependent graded expression of the target protein. The system is based on the E. coli arabinose operon encoding enzymes needed for catabolism of arabinose. - araC encodes the regulator AraC. Operator 1, operator 2, inducer 1 and inducer 2 and the promoter called pBAD. The controlling molecule is the AraC (regulator). When AraC inhibits the promoter - cannot transcribe the BAD gene. → add arabinose → transcription of BAD gene. arabinose is acting as an inducer (a positive regulator). no arabinose → no expression. arabinose present → expression. - Acts as a repressor and an inducer. Promoters that rely on positive control have lower background expression levels. Binding of arabinose to AraC changes its conformation and defines its ability to bind operator sites near araBAD promoter; this again determines binding of RNA polymerase to araBAD promoter and transcription of recombinant gene

Briefly discuss different framings or understandings of "the public"

The public is not one thing or one group. It is important to think of the public as citizens and not just consumers/laypeople (someone not trained, Laity)/stakeholders. There is a heterogeneous nature of the public. There are differences in age, gender, education and culture, and this has an effect on what type of response the public gives. Stirling describes three rationales of public engagement, instrumental, substantive and normative - The public as consumers. The technology appears as a range of products and engagement of consumers is typically promoted to either achieve acceptance of existing products or enable the development of acceptance of future products. A common aim is to avoid the type of opposition that rose against GMO, the knowledge was reliable in the scientific sense, but not socially robust. rational-instrumental - The public as a laity. This engagement is often represented as important because there is a need for members of the public to be educated and informed about the different subjects in question. This is to establish a relationship of trust between members of the public, scientists and decision makers. rational- instrumental - The public as stakeholders. The technology is portrayed as a development with socially transformative potential and involving risks that need to be managed to minimize harm. The aim is to ensure that development proceeds through careful consideration of the range of potential impact and interest involved. rational substantive. - The alternative approach, normative. Public needs to be framed as citizens for this rationale, people can express concerns outside of imposed categories. Individuals are empowered and acts as members of communities.

Give one example or reason why focusing only on risk and risk assessment often provide a too narrow approach when considering new and emerging technologies.

The risk assessment can come at expense of social, economic and political consideration. Spending too much time assessing and managing unlikely risks can divert resources that could be used more profitably. Much time have to go for training, the time spent for development and research will have to be used for training to ensure proper execution of risk management. Collingridge dilemma. Impacts of the technology cannot be predicted until the technology is developed and widely used, but control is difficult when technology has become entrenched

thermodynamics

Thermodynamics constrain reactions directionally. As different reaction have different thermodynamic characteristics that determine if the reactions are spontaneous or not.

Give at least one example of unintended consequences of socio-technical development and explain why this has had consequences for the relation between science and society.

Unintended consequences of developments can increase the gap between science and society. There will be a lack of trust in science and government. Mad Cow disease? GMO? Hydrogen bomb, Chernobyl. ● Increased gap between science and society ○ Unintended consequences, ex hydrogen bomb ● Lack of trust in science and governments ○ GMO controversy ■ Opposition rooted in skepticism about the framing of problems and solutions, not just emotions. ■ Skepticism about motivation of actors to employ biotechnology in agriculture ■ Emotions vs framing ● Public protests ● How should this be addressed?

How can we make microbiomes to perform specific chemical conversions in a wastewater treatment process?

We need to engineer environments with suitable selective forces, that provide ecological riches to the right ecological guilds. This requires a solid understanding of microbial ecophysiology and transport processes, and can be informed by -omics data