HL Paper 3
Beans contribute to flatulence. Alpha-galactosidase, derived from the fungus Aspergillus niger, is an enzyme that breaks down the fibre usually fermented by bacteria, reducing intestinal gas. Describe how alpha-galactosidase would be produced using A. niger in a continuous fermenter.
Temperature is a variable that needs to be continually monitored in deep-tank batch fermentation of penicillin. List two other variables that need to be monitored.
Plant-derived proteins are likely to be safer for human use than those derived from mammalian cell cultures, as plant pathogens are not harmful to humans. The hepatitis B vaccine has been produced in tobacco plants.
In an experiment, soybean (Glycine max) cells were transformed using Agrobacterium tumefaciens to produce hepatitis B surface antigen (HBsAg). The amount of HBsAg made by the transformed soybean cells was measured at different times after transformation. The results are shown in the bar chart.
[Source: Reprinted by permission from Springer Nature from Plant Cell Reports.
Analysis of the limitations of hepatitis B surface antigen expression in soybean cell suspension cultures.
Ganapathi, T.R., Sunil Kumar, G.B., Srinivas, L., Revathi, C.J. and Bapat, V.A., © 2007. https://doi.org/10.1007/s00299-007-0379-7.]
Describe how the tobacco mosaic virus is used in the production of hepatitis B vaccine.
Using the data, identify one limitation of using soybean cell cultures.
The open reading frame (ORF) of HBsAg used in tobacco plants was the same one used in soybean plants. Define ORF.
Describe one bioinformatic method that could have been used to find the gene sequence for HBsAg.
Transgenic rainbow trout (Oncorhynchus mykiss) were produced from both wild strain and domestic strain trout, using a gene coding for growth hormone from coho salmon (Oncorhynchus kisutch). The graph shows the mean mass of the nontransgenic and transgenic trout at 8 months post-fertilization.
[Source: Reprinted by permission from Macmillan Publishers Ltd: Nature, 409, Growth of domesticated transgenic fish, R H Devlin et al., pp. 781–782, copyright 2001]
Analyse the data for the growth of nontransgenic trout and transgenic trout.
Suggest a reason for the growth differences between the nontransgenic trout and transgenic trout.
Describe the use of marker genes in the development of transgenic organisms such as trout.
Outline the possible environmental impact associated with the accidental release of transgenic trout.
The diagram shows an industrial anaerobic fermenter.
State one fuel that can be produced in this fermenter.
Outline one variable that must be controlled in an industrial fermenter
Explain factors that affect the rate of activity of microorganisms in fermenters.
The diagram represents the cell walls of Gram-positive and Gram-negative bacteria.
Label the layers I, II and III.
The following base sequence represents part of a larger DNA molecule that is going to be analysed for the presence of open reading frames.
Explain how this DNA can have six possible reading frames.
State the type of codon that helps to identify open reading frames.
Once an open reading frame is identified, explain the steps researchers would follow to determine a potential function for that sequence.
The cladogram is based on a comparison of open reading frames in DNA taken from fungi. It is an example of how open reading frames can be used in phylogenetic studies.
Outline how open reading frames are identified in DNA.
Explain what the branching off points represent in the cladogram of these fungi.
There are several methods of introducing DNA into a cell in the laboratory. Outline the introduction of recombinant DNA in plant cell protoplasts.
Distinguish between batch fermentation and continuous fermentation.
Aspergillus niger is used to produce citric acid by continuous fermentation. Glucose is converted to pyruvate by glycolysis. Trehalose-6-phosphate normally inhibits hexokinase, an important enzyme in the glycolysis pathway.
Suggest how pathway engineering could be used to address this factor which reduces yields of citric acid.
The figure represents a section of DNA.
Identify the first triplet of nucleotides of each of the three reading frames in the 5’ to 3’ direction.
Open reading frames have start and stop codons. State one other characteristic of open reading frames.
Explain how marker genes are used in genetic modification.
State one physical method that could be used to introduce a gene into a plant.
Citric acid is produced on an industrial scale and global production is over 1.4 million tons
per year with a rising trend in demand.
State one industrial use of citric acid.
State the scientific name (binomial) of the microorganism usually used in this process.
Biopharming is the use of genetically modified plants or animals as a source of pharmaceutical products.
Outline how a named vector is used to introduce a new gene into a plant.
State the role of marker genes.
Discs were soaked in different antibiotics and were placed on a culture of Bacillus subtilis spread on sterile agar in a Petri dish. The Petri dish was left in an incubator, after which zones of inhibition were observed surrounding some of the discs. The photograph is to scale.
[Source: Tasha L. Sturm. 2009. Kirby-Bauer disk diffusion susceptibility test. Visual Resources. American Society for
Microbiology, Washington, DC. www.microbelibrary.org Accessed 29 September 2014]
Estimate the diameter of the zone of inhibition of chloramphenicol.
Distinguish between the action of tetracycline and penicillin on B. subtilis.
Suggest a reason for the result with disc X.
Explain how it could be determined that B. subtilis is a Gram-positive bacterium.
B. subtilis colonies form biofilms through quorum sensing. Define quorum sensing.
B. subtilis colonies form biofilms through quorum sensing. State three possible advantages to B. subtilis of forming a biofilm.
1.
2.
3.
A segment of DNA is shown. Determine a possible open reading frame (ORF) segment in the DNA segment by completing the table.
In gene research, outline the use of open reading frames.
In gene research, outline the use of gene knockout.
In gene research, outline the use of BLASTn.
Crop genetic engineering was performed to improve drought tolerance in tomato plants (Solanum lycopersicum) by adding a gene from an edible fungus (Flammulina velutipes). The cotyledons of tomato plants were cut and co-cultivated with Agrobacterium tumefaciens containing the transgenic Ti plasmid. Plates containing kanamycin were used to select for transgenic cotyledons. The graph shows concentrations of three constituents of the wax that coats wild type plants (control) and transgenic tomato plants.
Outline the use of kanamycin in the selection of transgenic cotyledons.
State how the sequence of the target gene from the fungus could be identified using a bioinformatics tool.
Suggest whether the results of this experiment show that these transgenic tomato plants are more resistant to drought.
Distinguish between the structure of Gram-positive and Gram-negative bacteria.
Golden rice (Oryza sativa, GR) is the generic name given to genetically modified rice that produces beta-carotene (provitamin A). Golden rice was created by transforming rice with the gene coding for the PSY protein (phytoene synthase) from daffodil (Narcissus pseudonarcissus) or from corn (Zea mays).
The picture shows variations of rice.
A White wild-type rice
B Yellow Golden rice expressing the gene coding for PSY from daffodil
C Orange Golden rice expressing the gene coding for PSY from corn
[Reprinted from TRENDS in Plant Science, 10(12), S. Al-Babili and P. Beyer, 'Golden Rice – five years on the road – five years to go?',
pp. 565--573, Copyright (2005), with permission from Elsevier. https://www.sciencedirect.com/journal/trends-in-plant-science]
Outline how scientists would determine whether the gene coding for PSY from daffodils has been taken up successfully by rice DNA.
Discuss whether production of Golden rice is an example of biopharming.
Agrobacterium tumefaciens was used in the production of Golden rice varieties. Explain how this bacterium is used to produce genetically modified crop plants.
A bioinformatics analysis was performed on the protein PSY transcribed from the gene from corn and from daffodil to obtain the sequence alignment.
On the alignment, identify the longest part of the sequence where the consecutive amino acids are the same.
BLASTp was used to obtain the alignment of the genes coding for PSY. Outline reasons for BLASTn not being suitable for obtaining this alignment.
In the alignment there are dashes (–) in some positions. Deduce what is indicated by these dashes.
The diagram shows an overview of the Henriksdal wastewater treatment process in Stockholm, Sweden.
State a group of organisms that will be active in the fermenter labelled X.
Deduce, with a reason, whether X is a continuous fermenter or a batch fermenter.
Probes are used to monitor significant variables within the fermenter. List three significant variables that should be monitored in the fermenter.
State the main component of biogas.
Inside Y there are rotating paddles. Outline two reasons for these paddles being needed.
The genetic code is the information encoded within the mRNA sequence that is translated into proteins by living cells. The codon table is shown.
The alignment was used to obtain a cladogram of these organisms.
State the bioinformatics tool used to obtain the alignment.
State the meaning of the dash (–) in the alignment.
(i) Identify the longest amino acid sequence where there are no differences amongst the five genera.
(ii) Suggest, with a reason, whether the DNA coding for the amino acid sequence identified in (c)(i) must be identical for the five genera.
Describe briefly how the cladogram was obtained.
Determine which two genera are most closely related according to their cytochrome c protein sequence.
Outline what is meant by the term genetic markers.
Outline two uses of genetic markers.
Evaluate the use of viral vectors in gene therapy.
Outline the use of microarrays to test for genetic disease.
Compounds containing the cyanide group (CN) are used to help extract gold from gold-containing rocks called ore. The process results in heaps of rocks that are contaminated with cyanide, a toxin that can inhibit cellular respiration. The bacterium Pseudomonas fluorescens degrades cyanide to ammonia (NH3 ), which is less toxic.
In an effort to explore the conditions that lead to maximum degradation of cyanide, researchers sprayed different samples of cyanide-processed ore with one of three solutions:
• a sterile solution
• a solution containing a culture of P. fluorescens
• a solution containing a culture of P. fluorescens and sucrose.
Outline the evidence that P. fluorescens can degrade the cyanide.
Suggest how the addition of sucrose promotes the degradation of cyanide.
With respect to the degradation of cyanide by P. fluorescens, explain what is meant by bioremediation.
Metabolites that indicate disease can be detected in urine. State a metabolite found in urine and the disease it could indicate.
Discuss the implications of biopharming using a specific example.
The diagram below represents a small-scale biogas fermenter.
[Source: © Science in Society. http://www.i-sis.org.uk/BiogasChina.php]
Suggest one material that could be loaded into the biogas fermenter from which biogas can be produced.
Identify the ideal temperature and oxygen conditions inside the fermenter for efficient biogas production.
Temperature:
Oxygen:
Distinguish between batch and continuous culture fermentation.
Explain how conditions in the fermenters are maintained to maximize penicillin production.
Explain two or more laboratory tests that can be used to detect the presence of specific pathogens in patients with an infectious disease.
Some halophilic bacteria biodegrade benzene, which makes them useful in treating oil spills as benzene is contained in crude oil. Investigators added cultures of the bacteria to benzene solutions at different salinities in containers. The amount of benzene remaining in the container was recorded once a week for two weeks.
[Source: adapted from C A Nicholson and B Z Fathepure, (2004),
Applied and Environmental Microbiology, 70 (2), pages 1222–1225]
Outline what is meant by halophilic when describing the bacteria.
Identify the salt concentration with the greatest rate of benzene biodegradation.
………………......… mol m–3
State one genus of halophilic bacteria known to biodegrade benzene.
Suggest one advantage to the bacteria of breaking down benzene.
Lipid A is a phospholipid that makes up the external layer of the outer membranes of most Gram-negative bacteria. LpxC is an enzyme involved in the biosynthesis of lipid A. In this experiment, a lawn of the Gram-negative bacterium Escherichia coli was grown on a nutrient agar plate. Shortly after inoculation, before the lawn is formed, discs containing different test compounds were placed on top. The Petri dish shows the results after 24 hours incubation.
Outline the effect of disc 3 on the bacterial lawn.
Outline the effect of mutating the LpxC inhibitor.
Predict the results obtained with disc 1 in a Gram-positive bacterial lawn.
Outline the characteristics which would indicate biofilm formation in Bacillus subtilis.
The formation of hydrophobic concrete is an example of how biofilms can be useful. Outline one example where biofilms can cause environmental problems.
Bacterial biofilms play an important role in urinary tract infections. They can be responsible for persistence of infections. The diagram shows biofilm formation.
Identify the step where the extracellular matrix first appears.
Explain the persistence of urinary tract infections, if bacterial biofilms are formed.
Escherichia coli, a Gram-negative bacterium, is a common cause of urinary tract infections. State the colour of E. coli bacteria after Gram staining.
Citric acid was produced in a fermenter using the pulp of cassava (Manihot esculenta) and the fungus Aspergillus niger. This process was carried out at room temperature for 6 days and citric acid was then collected. The apparatus was cleaned and set up for a new fermentation. The process is shown in the diagram.
[Source: Prado, F.C., Vandenberghe, L.P.S., Woiciechowski, A.L., Rodrígues-León, J.A. and Soccol,
C.R., 2005. Citric acid production by solid-state fermentation on a semi-pilot scale using different percentages of
treated cassava bagasse. Brazilian Journal of Chemical Engineering, 22(4), pp.547–555.]
State in which numbered part of the process you would find the probes to detect changes in pH.
Explain the possible causes of these changes in pH.
Suggest, with a reason, whether this is a batch or a continuous fermentation.
State one use of the citric acid produced.
A protein expressed by the gene carotene desaturase (CTP1) was introduced into Golden rice (Oryza sativa) through genetic engineering. Bioinformatics were used to determine if the protein sequence matched any allergenic proteins (proteins that trigger an allergic reaction). The table shows the results from the alignment of the CTP1 protein with proteins from moth, soybean and dust mite that are known to be allergenic.
State a bioinformatic search tool that could be used to perform the alignment.
Outline how the similar protein sequences were found.
Regulations say that a protein is considered allergenic and unsafe for human consumption if at least 80 amino acids are aligned and there is greater than 35 % identity with any known allergen. Analyse the data provided to consider whether CTP1 is safe for human consumption.
Genes such as the one coding for CTP1 can be located by searching for open reading frames. Outline how open reading frames are identified.
Physical and chemical methods can be used to genetically modify crop plants by inserting new genes. List a physical and a chemical method that could have been used to introduce the gene coding for CTP1 into the rice plants.
Some microorganisms can form a biofilm on living or non-living surfaces. The image shows a Staphylococcus aureus biofilm inside a medical catheter.
Define biofilm.
Explain the difficulties of treating microorganisms growing in biofilms.
Explain the use of a named bacterium in bioremediation.
Explain how BLAST searches are carried out and the applications of different types of these searches.
Disinfectants can be used to break up the biofilms. One of the most commonly used disinfectants is chlorine. Hypochlorous acid forms when chlorine mixes with water. Two different disinfectants were tested experimentally: monochloramine and hypochlorous acid. The concentration of disinfectant needed to kill 99 % of the bacteria was determined, both for free bacteria and for bacteria in biofilms. The table shows the results.
[Source: LeChevallier, M W et al. “Inactivation of biofilm bacteria.” Applied and environmental
microbiology vol. 54,10 (1988): 2492–9. doi:10.1128/AEM.54.10.2492-2499.1988 reproduced/amended with
permission from American Society for Microbiology.]
Bacteria sometimes form biofilms inside metal pipes in water systems. Distinguish between free bacteria and bacteria in biofilms.
The use of monochloramine is replacing the use of chlorine, as it is more stable, but it can produce by-products that pose possible health risks. Evaluate the data to see whether monochloramine is a good choice as a disinfectant for water systems.
State how viruses could be used to treat water systems, in order to avoid the use of a disinfectant.
Explain the process of gene therapy using viral vectors.
The table shows a comparison of DNA base sequences in several yeast (Saccharomyces) genomes.
[Source: P. F. Cliften et al. (2001) ‘Surveying Saccharomyces Genomes to Identify Functional Elements by Comparative DNA Sequence Analysis’, Genome Research, 11, pp. 1175–1186. © Cold Spring Harbor Laboratory Press. Reproduced with permission.]
Identify the species that has the lowest percentage of coding sequences.
State how similar nucleotide sequences can be identified.
The yeast Saccharomyces cerevisiae was the first eukaryotic organism to have its entire genome sequenced. Suggest reasons for the choice of yeast as a study organism.
Outline possible medical applications of the polymerase chain reaction (PCR).
Outline one way in which genetic sequences can be used to indicate predisposition to a disease.
Outline the use of luminescent probes in the treatment of tumours.
Corn (Zea mays) is by far the most widely used biopharming plant, followed by soybeans, tobacco and rice. Around the world approximately 400 biopharming products are in open-air field trials.
State one possible application of biopharming.
Explain the use of a viral vector in gene therapy.
Outline the main principles of the Enzyme-Linked Immunosorbent Assays (ELISA) test.
Outline one example of the use of a marker gene in genetic engineering.
The graph shows the trends of the use of genetically modified corn in the United States (USA) from 2000 to 2015, including herbicide tolerant varieties (HT), insect resistant varieties (Bt) and varieties with both traits combined.
Compare and contrast the use of genetically modified corn in the USA in the years 2000 and 2015.
Explain how the Bt and HT combined crop was produced.
The scanning electron micrograph shows a biofilm on a metal surface from an industrial water system.
[Source: Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms, Rodney M. Donlan, J. William Costerton, Clinical Microbiology Reviews, 2002, 15 (2), pp. 167–193. Reproduced with permission from American Society for Microbiology]
Outline the emergent properties of biofilms.
State a positive application of biofilms.
Suggest two problems that could be caused by the presence of biofilms in water systems.
The diagram shows the formation of a biofilm in a mammary gland, producing a mastitis infection.
[Source: © International Baccalaureate Organization 2019]
Outline the process of quorum sensing in bacteria forming a biofilm.
Suggest one reason, other than quorum sensing, for the resistance to antibiotics of a biofilm.
Outline one example of an environmental problem caused by biofilms.
Explain the use of DNA microarrays in genetic testing or diagnosis.
Explain how infection by a pathogen can be detected by the presence of its genetic material and of its proteins.
The diagram shows a spherical array of phospholipid molecules enclosing a water droplet. Such structures can be used to introduce genes into plant protoplasts.
[Source: SuperManu, https://en.wikipedia.org/wiki/Liposome#/media/File:Liposome_scheme-en.svg]
Explain briefly how plant protoplasts are prepared and how vesicles can be used to introduce genes into them.
The micrograph below shows an example of a biofilm including Staphylococcus aureus.
Biofilms can be formed in many different environments.
State one example of an environment where biofilms can be formed.
Discuss the emergent properties of biofilms.
Cupriavidus metallidurans CH34 is a heavy metal-resistant bacterium that was genetically modified to be used for bioremediation. The merB gene that controls the conversion of organic mercury into inorganic mercury was introduced in the bacterium. The gel electrophoresis image below shows the presence of the merB gene in two strains after 70 generations.
[Source: Copyright © 2011 Rojas LA, Yáñez C, González M, Lobos S, Smalla K, Seeger M (2011) Characterization of the
Metabolically Modified Heavy Metal-Resistant Cupriavidus metallidurans Strain MSR33 Generated for Mercury Bioremediation.
PLoS ONE 6(3): e17555. https://doi.org/10.1371/journal.pone.0017555]
Outline the aims and methods of bioremediation.
The generation time of C. metallidurans is a few hours. Two strains of the bacterium were tested for the presence of the merB gene 70 generations after the genetic modification. Suggest one reason for carrying out these tests after 70 generations of the transgenic bacterium.
Explain the use of Pseudomonas in bioremediation.
Caenorhabditis elegans, a nematode, was the first multicellular organism whose genome was completely sequenced.
Outline the benefits of using model organisms for studying gene function.
Describe how BLAST can be used to establish phylogenetic relationships between several organisms.
A DNA microarray chip was prepared as a collection of microscopic DNA strands from human genes attached to a solid surface. The diagram shows part of the microarray chip after hybridization with cDNA produced from normal cells and from cancer cells. cDNA of normal cells was labelled with green fluorescent dye and cDNA from cancer cells was labelled with red fluorescent dye. Both sets of labelled cDNA were then allowed to bind to the microarray.
Explain the reason that only cDNA from expressed genes binds to the DNA on the chip.
Explain how the information obtained in this microarray accounts for the differences between normal cells and cancer cells.
Explain how antithrombin can be produced by biopharming.
Discuss biopharming.
Discuss the use of microorganisms in bioremediation.
Discuss the use of microarrays in the diagnosis of disease.
Explain the formation of biofilms and the problems associated with their formation.