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.

a. constant nutrient medium «supply» needed/maintained

b. optimal mixing

c. fermented in sterile bioreactor

d. alpha-galactosidase production/general conditions assayed/screened/monitored «throughout the process»

e. continuous removal of alpha-galactosidase/products

[Max 3 Marks]

a. pH

b. «dissolved» oxygen

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.

a. fusion between genes of (viral) capsid and hepatitis B antigen/HBsAg;

b. (transformed) virus infects tobacco plant;

c. tobacco plant expresses antigen/HBsAg;

d. HBsAg producing plants fed to animals;

e. animals produce anti-HBsAg antibodies;

as time passes, the soybean produces less antigen / stabilizes to a lower level (after 6 months)

DNA/nucleotide sequence with a start codon and stop codon coding for a polypeptide chain;

a. gene databank; (e.g. GenBank/NCBI);

b. BLASTn to search similar DNA sequences;

c. ORF finder to search for the start codon;

Alternative:
d. protein database search for antigen (sequence);

e. example of database; (e.g. NCBI/SwissPro/Uniprot/PDB/other checked database);

f. BLASTp to search similar protein sequences;

Most have some knowledge of the usage of the tobacco mosaic virus but had difficulty in providing clear answers.

Most candidates correctly identified that the effect is reduced over time.

It was difficult for many candidates to specify that a start and a stop codon were necessary in the definition of an open reading frame.

Most answers didn't contain enough elements, although many mentioned BLASTn.

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.

a. both transgenic «strains» show more growth/mean mass than nontransgenic

    Allow vice versa

b. wild nontransgenic «strain» showed less growth than wild transgenic

    Allow vice versa

    OR

    greatest difference between wild nontransgenic and transgenic «strains»

    OR

    wild «strain» showed less growth/mean mass in nontransgenic but reverse in transgenic

c. mean mass/growth in domestic nontransgenic «strain» lower than «domestic»
transgenic

d. error bars overlap for domestic nontransgenic and transgenic «strains»

[Max 2 Marks]

gene for growth hormone has been assimilated/is expressed in the transgenic trout

OR

more growth hormone produced/expressed in transgenic trout

a. indicates successful uptake of recombinant DNA

b. identifies transgenic organisms

c. example of a marker gene

eg: antibiotic resistance gene in bacteria

[Max 2 Marks]

a. transgenes may be transferred to other species/organisms

b. may alter ecosystem/food chain

c. may outgrow other species

    OR

    decrease biodiversity

    OR

    outcompete nontransgenic individuals/trout

[Max 2 Marks]

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.

methane / biogas

a. anaerobic conditions / prevent entry of oxygen 

b. warm temperatures / temperature above 15°C 

c. different communities of microorganisms / Eubacteria and methanogens 

d. proper pH needs to be maintained

a. changes of pH (possibly caused by changes of CO₂) affect enzyme activity 

b. ambient pressure / build-up of gases may affect reactions involving gases 

c. increase of temperature could affect reaction rates /denature enzymes 

d. decrease in nutrients/raw materials/change in substrate could decrease rate of product formation

e. increase in wastes could decrease rate of metabolism 

f. particle size affects rate of fermentation associated with surface area (for substrate interactions) 

g. mixing is necessary as sedimentation lowers surface area thus decreasing rate of reactions

l. peptidoglycan 

ll. outer membrane/layer of lipopolysaccharide and protein 

lll. peptidoglycan

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.

a. «three» reading frames can occur in either strand 

b. from 5' « to 3' » 

c. reading frame can start from any of the first three nucleotides 

d. from the top strand: GTG or TGA or GAA as first triplet  OWTTE
OR
from the bottom strand: ATA or TAT or ATT as first triplet

start codon/AUG
OR
stop codon/UAA/UAG/UGA

a. use a database 

b. conduct BLAST search
OR
BLASTn allows DNA sequence alignment 

c. «sequence alignment software used» to identify/compare similar sequences in different organisms 

d. gene function can be studied using model organisms with similar sequences with known function OWTTE

e. BLASTp allows protein alignment
OR
EST may be used to identify gene activity 

f. can change sequence and create “knockout” study organism 

g. changes in phenotype due to knockout procedure allow determination of function 

h. valid example provided

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.

a. identify a start codon and stop codon 

b. identify base sequences for a gene/that could code for a polypeptide 

c. possible correlation with existing open reading frames in databases

a. represent common ancestors shared by the organisms that emanate from the point 

b. indicates time since divergence 

c. indicates number of differences in DNA

a. plant cells made into protoplasts by removing their cell wall / use cellulase to produce protoplasts

b. physical methods such as electroporation /microinjection/biolistics

c. chemical methods such as liposomes/calcium chloride/polyethylene glycol «PEG»

d. vectors such as Agrobacterium/tobacco mosaic virus

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.

a. «genetically modify to» incorporate gene for low/blockage of TPS activity into A. niger 

b. «genetically modify to» incorporate gene that breaks down trehalose-6-phosphate 

c. selectively breed A. niger cultures for low/no TPS activity

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.

«frame 1 =» ATG

«frame 2 =» TGC ✔

«frame 3 =» GCC

All three required.
Order of frames is not relevant.
Do not accept RNA, e.g. AUG.

continuous stretch of codons «between start and stop codon»
OR
can contain introns
OR
sufficient nucleotides to code for a polypeptide chain ✔

a. «marker genes» are attached to target genes and inserted into bacteria/host cells ✔ OWTTE

b. «marker genes» confirm that the target gene has been successfully integrated into the genome/ plasmid ✔

c. «marker genes» for antibiotic resistance/fluorescence ✔

d. antibiotic is added to the media used to grow the bacteria
OR
UV light is used «to detect fluorescence» ✔

e. genetically modified bacteria will survive
OR
successful transgenic organisms will fluoresce
OR
allow researchers to identify the bacteria that have been modified ✔

electroporation
OR
microinjection
OR
biolistics/gene gun/gunshot ✔

State one industrial use of citric acid.

State the scientific name (binomial) of the microorganism usually used in this process.

flavour enhancer/«food» preservative ✔

Aspergillus niger ✔

Both names in full required.

Candidates did generally well in all parts of this question.

Candidates did generally well in all parts of this question.

Outline how a named vector is used to introduce a new gene into a plant.

State the role of marker genes.

ALTERNATIVE 1
a. Agrobacterium tumefaciens contains a tumour-inducing/Ti plasmid ✔

b. required/target gene inserted into plasmid ✔

c. bacterium injects modified plasmid into plant cell and DNA becomes incorporated into plant cell nucleus ✔

ALTERNATIVE 2
d. tobacco mosaic virus/TMV is the vector ✔

e. required/target gene inserted into TMV ✔

f. TMV injects modified DNA into plant cell «and DNA becomes incorporated into plant cell nucleus» ✔

to verify that the target gene has been incorporated in the target cell/organism ✔

OWTTE

Most had some knowledge about the usage of vectors to introduce genes, but their difficulty was in structuring their answer.

Most could state the role of marker genes.

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.

16 mm or 1.6 cm ✔

units required

Allow 15 mm to 17 mm, or 1.5 cm to 1.7 cm

tetracycline was more effective in inhibiting/killing the bacteria ✔

a. it contained no antibiotic ✔

b. it was a control «with only water» ✔ 

c. the antibiotic it contained was not effective against the bacterium/bacterium is resistant to antibiotic ✔

a. use the Gram staining procedure ✔

b. Gram-positive bacteria take up/retain «crystal violet» stain ✔ 

c. «Gram-positive bacteria» appear purple-coloured seen through a microscope ✔

«a system of» behaviours triggered as a function of population density ✔

a. allows a broader range of habitat «for colonization» ✔

b. biofilms are resistant to physical forces/heat shock/desiccation/physical wiping ✔

c. biofilms can withstand nutrient deprivation ✔

d. resistance to changes in pH ✔

e. resistance to antibiotics ✔

f. avoid phagocytosis ✔

Although this question was correctly answered by the majority of students, there were students who found it difficult, possibly because they did not bring a ruler to the examination. Students should also be reminded to use SI units only. Measurements in inches are not acceptable.

Students had no trouble identifying tetracycline as being more effective.

This question was also well answered, with most students recognising the disc as a control or the fact that the bacteria were resistant. Weaker students struggled with subject-specific terminology and mistakenly used terms such as “antibodies” (instead of “antibiotics”) and “immune” (instead of “resistant”).

Most candidates who attempted this option were familiar enough with the technique to score at least 1 mark.

This question was a good discriminator, as weaker students were unable to use correct terminology such as “population density,” instead resorting to vague phrases like “when the bacteria sense something.”

For two years in a row, option B questions about biofilms have been well answered. The most commonly identified advantages of forming a biofilm were resistance to antibiotics and resistance to physical forces, such as wiping.

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.

a. correct starting point ✔
eg

OR

b. correct RNA nucleotides ✔

identify a sequence/gene «that has the potential to be transcribed» ✔

silence gene to observe the effect when the gene is not expressed
OR
change in phenotype to deduce function of gene ✔

to compare nucleotide/DNA sequence with other «nucleotide» sequences ✔

Most candidates had difficulty completing the diagram and/or used DNA nucleotides instead of RNA nucleotides.

Knowledge of different tools in gene research seems to be fine, but gene knockout was less understood.

Knowledge of different tools in gene research seems to be fine, but gene knockout was less understood.

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.

a. kanamycin resistance as marker gene

b. when organisms grown in kanamycin, only resistant survive
OR
those that took up resistance/cloned ones survive

database/NCBI search to find target gene/OWTTE
OR
search for target gene in other/related organisms

Allow other named database.
Please check unfamiliar names for authenticity.

a. more wax deposition constituents «in leaves» of transgenic than control plants

b. wax is waterproof

c. less evaporation from «waxy» leaves «protects from drought»

Distinguish between the structure of Gram-positive and Gram-negative bacteria. 

a. Gram-negative bacteria have a thinner peptidoglycan cell wall / Gram-positive bacteria have a thicker peptidoglycan cell wall 

b. Gram-negative bacteria have an additional membrane of «lipopolysaccharide and protein» outside the wall «whereas Gram-positive bacteria do not»

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.

a. by detecting a marker/resistance/sequencing gene ✔
Accept PCR to detect the marker gene.

b. the offspring produce yellow rice ✔

a. biopharming is the introduction of genes into another species for medical use ✔

b. β-carotene/provitamin A is added to rice to prevent «night» blindness/diseases ✔

c. through genetic engineering ✔

a. «Ti/tumour inducing» plasmid of A. tumefaciens/bacterium causes tumours/galls ✔

b. Ti incorporates genes «of β-carotene synthesis»
OR
Ti is used as a vector of the gene «for β-carotene» ✔

c. recombinant plasmids reintroduced into A. tumefaciens/bacterial cells ✔

d. bacteria infect rice plant cells ✔

e. the newly incorporated gene produces β-carotene/Golden Rice ✔

sequence shown on alignment:

a. BLASTp aligns amino acids «of proteins» ✔

b. BLASTn aligns nucleotides «of DNA/RNA» ✔

c. amino acid sequence is shown therefore BLASTn would not work ✔

a. dashes are shown where there is no alignment ✔

b. due to lack of amino acids on one of the sequences ✔

c. in order to make them align the computer introduces gaps ✔

d. the sequence with dashes does not include the sequence of the other protein shown ✔

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.

bacteria
OR
archaeans
OR
methanogens ✔

Accept a named bacterium

continuous because there is a constant input of raw materials ✔

a. pH/acidity/alkalinity ✔

b. foam ✔

c. metabolites ✔

d. substrate ✔

e. oxygen ✔

f. carbon dioxide ✔

g. temperature ✔

h. pressure ✔

Accept other valid condition

methane ✔

a. mixing substrate to increase contact with bacteria
OR
preventing sedimentation ✔

b. avoids formation of biofilms ✔

c. aeration for aerobic bacteria ✔

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.

BLASTp

Do not allow “BLAST” alone but accept BLASTx.

gap/no amino acid present «for cytochrome c in that organism in that position»
OR
protein is shorter

(i) GLFGR

Can be shown directly on the alignment.

(ii) no, because more than one codon can code for an amino acid/degeneration of the genetic code

ALTERNATIVE 1
a. alignment used to quantify differences and similarities

b. algorithms for cladograms
OR
named algorithms

Named algorithms: least squares, neighbour-joining, parsimony, maximum likelihood or Bayesian inference.
Allow other verifiable algorithms.

c. selection of best model

ALTERNATIVE 2
d. comparing amino acid sequences between organisms

e. more similar sequences correspond to closer evolutionary links/OWTTE
OR
number of differences indicate number of mutations accumulated «over time»
OR
«if mutation rate is assumed to be constant», more mutations imply further evolutionary distance

f. length of lines/position of nodes established by the number of differences/mutations between organisms

Marks should be awarded for statements from only one alternative, not both.

Zea «corn» and Oryza «rice»

Both required.

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.

A gene/DNA sequence «with a known location on chromosome» used for identification

a. to identify species/pathogenic organisms
OR
successful uptake of DNA in genetically modified organisms/GMOs 

b. to detect disease due to variation in DNA «substitution/deletion» 

c. to determine risk of developing certain disorders 

d. to confer resistance to antibiotic/agent that would normally kill it 

e. to make cells containing gene look different
OR
green fluorescent tag makes cells visible under UV light

a. gene therapy trials have used viruses to deliver un-mutated copies of genes to the «somatic» cells of the patient's body 

b. examples of the use of viral vectors  eg gene therapy may provide a way to cure genetic disorders, such as severe combined immunodeficiency

c. one of the main problems is immune response to viruses / may cause toxicity/disease 

d. some viral vectors insert their genomes at a random location on one of the host chromosomes «which can disturb the function of cellular gene» / enter wrong cells «if targeting tumour» / could lead to cancer

a. analyze tissue/blood sample for DNA sequence 

b. each spot «on microarray» has small quantity of specific DNA sequence/probe 

c. reverse transcriptase used to make cDNA 

d. fluorescent dye linked to cDNA 

e. «cDNA» binds to/hybridizes with probes that have complementary base sequences 

f. fluorescence/different colours shows probes have hybridized / which sequences were in the tissue sample

Allow specific examples of genetic diseases.

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.

a. in sterile solution/control there is no degradation of cyanide but there is in the solutions with P. fluorescens 
OWTTE

b. in solution containing P. fluorescens and sucrose degradation of cyanide higher than without sucrose 

c. control with sucrose «only» missing to establish causality OWTTE

«organic» carbon source «necessary for the reaction to degrade cyanide»
OR
sucrose provides the energy source

a. bioremediation is the use of organisms to degrade pollution/toxins in the environment 

b. P. fluorescens necessary to degrade cyanide which is toxic to the environment OWTTE

c. often involves supplementing with nutrients/carbon source/aeration

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.

a. named metabolite

eg: glucose

b. associated disease

eg: diabetes

a. production of pharmaceuticals
OR
named example of biopharming

b. easily scaled to cover demands
OR
cheaper

c. drugs can be delivered in food «making it more attractive/easier to eat»

d. unethical/ethical aspect/OWTTE

e. allergic reactions/ side effects

f. horizontal gene transfer consideration

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.

blackwater
OR
brownwater/sewage
OR
feces
OR
organic solid waste
OR
paper pulp
OR
other biodegradable material ✔

Do not accept methane and CO₂.

temperature: warm
oxygen: low/anaerobic ✔

Both required.

a. in batch culture product is withdrawn at the end of the «organisms » growth phases whereas in continuous culture product is withdrawn while the organism is growing / OWTTE ✔

b. in batch culture all of the growth medium/nutrients are added at the beginning whereas in continuous culture, growth medium/nutrients are added at a constant rate / OWTTE ✔

c. batch culture is carried out in large scale fermenters whereas continuous culture could be in small scale ✔

d. continuous can run for a longer time than batch ✔

a. aerobic conditions achieved by stirring/aeration ✔

b. buffers maintain pH ✔

c. moderate temperatures maintained by cooling jacket ✔ Accept mention of 20-30°C.

d. product is collected during stationary phase ✔

Award marks for identification of the factor and description of the effect.

Explain two or more laboratory tests that can be used to detect the presence of specific pathogens in patients with an infectious disease.

a. blood/urine cultures to diagnose bacterial infections ✔

ELISA:

b. «ELISA» uses antibodies specific to pathogen antigen ✔

c. the antibodies are linked to an enzyme ✔

d. after binding of the antibody enzyme complex samples are washed to remove unbound complex ✔

e. is added for the enzyme which changes colour «if they join with an antigen» ✔

MICROARRAY:

f. test for specific mRNA sequences «using a microtiter plate» ✔

g. reverse transcriptase used to make cDNA from mRNA, linked with fluorescent dye ✔

h. «laser» light detects when cDNA and DNA hybridize which confirms presence of protein ✔

PCR:

i. detection of genetic material from the pathogen is obtained using a PCR «by using primers based on pathogen sequences» ✔

j. if DNA/RNA is amplified «more than control» then infection is confirmed ✔

k. problems of false positive or false negative «in test result interpretation» ✔

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.

thrive/live in a salt rich environment ✔

1 mol m^–3 ✔

Marinobacter ✔

Allow other correct responses eg: Dechloromonas, Haloferax, Pseudomonas, Fusarium, Halomonas, Halococcus,
Halobacterium, Haloarcula, Haloarchaea, Fundibacter, Fusarium, Alcanivorax, Dietzia

a. energy gained from the benzene ✔ 

b. benzene acts as a source of carbon ✔

(a) to (c) These three questions were often answered well.

(a) to (c) These three questions were often answered well.

(a) to (c) These three questions were often answered well.

Very few students could state that the breakdown of benzene could be used as a source of carbon or energy.

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.

a. the antibiotic/ampicillin diffuses out

b. killing bacteria/inhibiting growth of bacteria

c. zone of inhibition/clearing formed

lipid A production/synthesis is not inhibited so bacteria can grow
OR
bacteria grow/are not affected since inhibitor function is lost

no inhibition of growth, since Gram-positive do not have lipid A in membrane/OWTTE

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.

a. bacteria/B. subtilis adhere to surfaces ✔

b. «bacteria/B. subtilis» grow surrounded by EPS/exopolysaccharide/extracellular polymeric substance matrix ✔

c. «bacteria/B. subtilis growing in a biofilm» are highly resistant to antimicrobial agents ✔

d. «bacteria/B. subtilis in biofilms» cooperate through quorum sensing ✔

a. example of where biofilm cause problems ✔
eg: clogging/corrosion of pipes/water systems ✔
slows down/interrupts/clogs water supply systems ✔
OR
eg: contamination of surfaces in food production ✔
causes food transmitted diseases ✔
OR
eg: plaque formation on teeth ✔
causes cavities ✔
OR
eg: catheter clogging ✔
causes urinary infections ✔

b. description of problems ✔

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.

ll

a. extracellular matrix does not let antibiotic enter
OR
has physical properties/adherence that make it hard to remove 

b. emergent properties may spread/cause antibiotic resistance 

c. quorum sensing communicates/signals to all cells 

d. slow reproduction rate/growth makes antibiotic less effective
OR
dormant cells have lower metabolic rate so antibiotic not that effective 

e. some bacteria escape the biofilm to colonize other areas/invade «renal» tissue

pink/red

Do not accept purple or violet.

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.

2

a. pH decreased by citric acid;

b. (pH lowered by) production of CO₂ produced (by the fungus) in respiration;

c. (pH lowered by) production of carbonic acid by CO₂ dissolving in water;

batch fermentation because the citric acid is collected at the end of the fermentation
OR
batch fermentation because the process was carried out over 6 days then re-set up
OR
diagram does not show constant supply of nutrients so it must be a batch;

preservative/flavour

Accept other verified use, e.g. buffering.

Most could correctly identify part 2, others wrote part 1.

Most stated that the pH changes were due to acidic conditions, but fewer could explain how the acidic conditions were generated.

Most suggested that it was a batch fermentation, but some omitted to provide a reason; a few others had incorrect answers.

Good candidates correctly stated flavour enhancer, others didn't know.

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.

BLASTp
OR
ClustalW
OR
FASTA3

database/named database searched to compare newly identified sequences with sequences of known function in other organisms

a. dust mite protein has 45% identity with CTP1 which could be unsafe 

b. only 36 aligned amino acids of dust mite protein which is less than the 80 danger limit 

c. both moth and soybean protein have less than 35% identity so are considered safe 

d. both moth and soybean protein have more than 80 amino acids aligned so could be unsafe 

e. CTP1 is not an allergen as it does not fit the criteria for any allergen
OR
CTP1 from the three organisms is not over the safety limit in the two categories so are considered safe

a. a DNA sequence with a promoter, a start and a stop codon 

b. start codon is ATG 

c. stop codon is TAA/TAG/TGA 

d. nucleotide sequence with a considerable length 

e. named bioinformatics software
OR
ORF finder

Allow other verifiable answer.

a. physical:
electroporation
OR
microinjection
OR
biolistics «gunshot» 

Allow other verifiable answer.

b. chemical:
calcium chloride
OR
liposomes

Allow other verifiable answer.

Define biofilm.

Explain the difficulties of treating microorganisms growing in biofilms.

Explain the use of a named bacterium in bioremediation.

«a biofilm is» any group of microorganisms in which cells stick to each other on a surface

OR

«a biofilm is» any group of microorganisms in which cells cooperatively produce a polysaccharide matrix

a. biofilms show properties that are not shown by the individual cells / biofilms develop emergent properties 

b. quorum sensing changes microbial behaviour/production of matrix 

c. (matrix) hard to remove from a surface 

d. biofilms are tolerant to desiccation and heat shock 

e. biofilms may be very resistant to the action of (many) antibiotics/antimicrobial agents 

f. because cell division in biofilms is slow, antibiotics targeting this will be ineffective 

OR

matrix forms a barrier to antibiotics

a. (bioremediation is the) use of microorganisms to consume/break down pollutants 

b. name of bacterium (genus required) and contaminating substance 

c. outline one action of (this) bacterium to remove environmental contaminants from water or soil 

d. another detail of use of (this) bacterium to remove the contaminant 

e. advantage/disadvantage of using this bacterium

b. e.g. some Pseudomonas to remove oil spills 

c. uses crude oil for energy / breaks down crude oil 

d. e.g. bacteria also need other chemicals/process may be speeded up by adding essential inorganic nutrients (phosphates/nitrates) 

OR

b. e.g. Pseudomonas used to remove methyl mercury

c. bacteria (Pseudomonas) degrade methyl mercury into elemental mercury and methane 

d. methyl mercury is very toxic / can be biomagnified in food chain

Explain how BLAST searches are carried out and the applications of different types of these searches.

Process (max [5]):

a. BLAST «Basic Local Alignment Search Tool» search enables comparison of an unknown sequence with databases of sequences 

b. «software» finds similar sequences / aligns sequences by locating matches between two sequences 

c. carries out statistical calculations «to find matches with other sequences» 

d. BLASTn used to align/show similarities in nucleotide sequences in nucleic acids 

e. BLASTp used to align/show similarities in amino acid sequences in proteins 

f. used to identify the gene of a protein 

Application (max [2]):

g. one application of BLAST 

h. second application of BLAST

eg BLAST can be used for identifying species / locating domains / establishing phylogeny / DNA mapping / other verifiable examples

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.

a. biofilm bacteria are all together while free bacteria do not interact with others;

b. biofilm bacteria present emergent properties not present in free bacteria;

c. quorum sensing only found in biofilm;

d. EPS matrix only in biofilm;

e. biofilm bacteria are more resistant to antibiotics/disinfectants;

Monochloramine not a good choice [1 max]
a. high concentrations/amounts are needed;

b. health risks need to be assessed;

Monochloramine a good choice [1 max]
c. monochloramine is more stable;

d. monochloramine range for biofilm is very extensive
OR
some biofilm bacteria must be highly resistant;

viruses/(bacterio)phages that are specific to the bacterium are used to kill it;

Good candidates managed to distinguish between free bacteria and bacteria in biofilms.

Most candidates stated that monochloramine was not a good choice as a large concentration is required, which could imply health risks.

Most knew that viruses were used but could not specify that they were specific to the biofilm bacteria; few stated that they were bacteriophages.

Explain the process of gene therapy using viral vectors.

a. used on genetic diseases caused by defective genes/lack of enzyme/ protein 

b. viral vector (genetically) modified for (safe) use 

c. virus genome is altered and missing gene/allele is introduced into virus 

d. a retrovirus is used to introduce RNA and reverse transcriptase in a host cell 

e. RNA is copied into DNA and introduced (permanently) into the cells genome 

f. could use adenovirus/virus with DNA to introduce genetic material 

g. in this technique DNA is not attached to cell genome 

h. thus not replicated during host cell replication / treatment has to be repeated 

i. it could be done in somatic treatment (body cells) or in sex cells (egg cells) 

j. example (ie Gendicine, is an adenovirus used for treatment of head and neck cancer)

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).

S. unisporus

BLASTn/sequence alignment software

“n” required in BLASTn

a. easy to grow

    OR

    easy/cheap to produce large amounts

    OR

    fast generation time

b. genomes are small/easy to manipulate

c. metabolically diverse

d. industrial applications/biopharming

e. no ethical issues «with yeast»

[Max 3 Marks]

a. identify different viral/influenza strains

b. genetic testing/testing for genetic disease mutations

c. tissue typing

d. vaccine development

[Max 1 Mark]

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.

a. genetic markers/specific sequences can be present in people with a disease  OWTTE

b. presence «of markers/specific sequences» indicates risk/probability of onset of condition Allow vice versa.

c. technique to detect the presence of the sequence  eg: PCR, electrophoresis, DNA sequencing, FISH, DNA databases, etc.

d. example of predisposition eg: BRCA sequence mutations indicating predisposition to breast cancer

a. transferrin/other protein taken up at higher rates by tumour cells 

b. transferrin/other protein can be labelled with a luminescent dye 

c. different tumour cell types can be distinguished/labelled in different colours 

d. can be used to highlight tumours «during surgery» OWTTE

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.

production of pharmaceuticals
OR
proteins used in therapy
OR
antibodies
OR
vaccines ✔

a. gene therapy involves introducing a normal copy of a defective gene into an organism ✔

b. virus/viral vector «genetically» modified for «safe» use ✔

c. insertion of desired gene/allele into viral genome/retrovirus ✔

d. an example is the use in SCID/lack of enzyme ADA in SCID ✔

e. removal of somatic cells ✔

f. introduction and insertion of the desired gene into the target cell
OR
modified virus can be introduced in the form of «inhaled» droplets
OR
the cells are introduced in the patient so that the desired gene can be expressed ✔

a. «ELISA» uses antibodies to detect a target antigen «to a pathogen» ✔ OWTTE

b. antigen attached to substrate ✔

c. the antibody «if present» will attach to the antigen ✔

d. «suitable» enzyme attached to antibody ✔

e. «enzyme» colour reaction shows the presence of antibodies to the antigen/pathogen ✔

Outline one example of the use of a marker gene in genetic engineering.

a. marker gene inserted into DNA containing target gene 

b. recombinant DNA «with marker gene and target gene» inserted into cell/organism 

c. named example of marker and target gene  eg: ampicillin resistance with BT gene for glyphosate resistance

d. further details of how the marker gene works eg: culture cells in ampicillin and if the cell grows into a callus, uptake has occurred

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.

a. large increase in (percentage of area planted with) genetically modified corn 

b. Bt and HT together hardly/not planted in the year 2000 but is majority of corn planted in 2015 

c. HT alone has slightly increased while Bt alone has decreased 

d. all 3 types of corn were planted in both 2000 and 2015

a. insect-resistant crops contain genes from the soil bacterium (Bacillus thuringiensis) Bt 

b. herbicide (glyphosate) resistance gene has been introduced in plants 

c. (resistance genes introduced) by vectors / physical methods / chemical methods 

d. successfully modified plants are selected for further propagation 

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.

a. properties not present in individuals but present/develop only in the aggregate

    OWTTE

b. develop structure/architecture/scaffolding

    OR

    develop an «extracellular» matrix/EPS

c. signaling/communication

d. migration/movement

e. resistant to antimicrobial agents

f. cooperates through quorum sensing

[Max 3 Marks]

a. sewage/waste water treatment/trickle filter beds

b. «bio»remediation of contaminated soil/water

c. metal extraction from ore deposits/microbial leaching

Accept other valid positive application

eg: decay/breakdown contaminants, such as petroleum

[Max 1 Mark]

a. contamination/pollution «of water system»

    OR

    «microbial growth of biofilm» causes disease through water systems

b. difficult to eliminate «from water systems»

c. fouling/clogging of water pipes

d. corrosion of water pipes

OWTTE

[Max 2 Marks]

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.

a. cells/bacteria in a biofilm are close together ✔

b. cells secrete signaling molecules ✔

c. «signaling molecules» bind to receptors of other cells
OR
«signaling molecules» allow communication between cells ✔

d. a threshold is reached which enables emergent properties ✔

a. polysaccharide matrix/EPS does not let antibiotic pass/limits transport of antibiotic ✔ OWTTE

b. reduced metabolic activity/growth rate of bacteria in biofilm contributes to resistance ✔

c. increased cell density limits transport of antibiotic «to the interior of biofilm» ✔

d. «horizontal» transfer of antibiotic resistance via plasmids ✔

a. area where biofilm problem exists ✔ eg emptying introduces invasive bacterial species into coastal waters.

b. environmental concern ✔

Allow other verified examples

Most candidates seem to have a very good knowledge of biofilms, but it was more difficult to outline quorum sensing properly and suggest a reason for antibiotic resistance.

Most candidates seem to have a very good knowledge of biofilms, but it was more difficult to outline quorum sensing properly and suggest a reason for antibiotic resistance.

Most could outline an example of biofilms but they had more difficulty relating it with an environmental concern.

Explain the use of DNA microarrays in genetic testing or diagnosis.

a. used to test the presence or absence or a gene/allele
OR
used to diagnose the presence of a particular strain of virus/bacteria
OR
used to diagnose the presence of a specific mutation
OR
used to diagnose the presence of an expression of genes ✔

b. DNA spots/probes/sequences attached to solid surface/microarray ✔

c. mRNA from healthy tissue/cell isolated and converted to cDNA
OR
mRNA from cancer tissue/cell isolated and converted to cDNA ✔

d. conversion to cDNA by reverse transcriptase ✔

e. fluorescent dye linked to complementary DNA/cDNA ✔

f. cancer cDNA colored with a different dye from the healthy cDNA ✔ Accept named colour.

g. cDNA binds to/hybridizes with probes «that have complementary base sequences» ✔

h. microarray rinsed to remove cDNA that has not hybridized ✔

i. microarray exposed to laser light which causes dye to give off light ✔

j. fluorescence shows which probes have hybridized
OR
fluorescence shows which sequences were in the tissue/sample ✔

k. hybridized probe shows gene expression
OR
hybridized probe helps in diagnosis of disease ✔

Explain how infection by a pathogen can be detected by the presence of its genetic material and of its proteins.

Presence of genetic material (PCR and/or microarray): [Allow 4 max]

PCR:
a. presence of the genetic material can be amplified by PCR;

b. if the genetic material is RNA, a reverse transcription PCR must be performed
OR
using reverse transcriptase, the enzyme must first transform RNA into DNA;

c. PCR primers stick to DNA;

d. amplify DNA;

Microarray:
e. genetic material can be detected in microarray;

f. probes of the DNA of the pathogen are placed on the chip;

g. if the pathogen DNA is complementary, there is fluorescence;

Presence of proteins: [Allow 4 max]
h. presence of the proteins can be detected by ELISA;

i. the test detects the antigens which are proteins of the pathogens;

j. it uses antibodies stuck to a plate (which capture the pathogen antigen)
OR
it detects antibodies against pathogen;

k. antibodies with an enzyme/fluorescence are used to reveal binding;

l. metabolites of the disease detected in blood/urine;

Only a few complete and detailed answers were provided by good candidates; most answers were based on previous examination sessions and mentioned some valid techniques, but failed to address the detection of the pathogen's genetic material on one hand, and its proteins on the other hand, which were specific to this session's question.

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.

a. enzymatically remove wall from plant cell to make protoplast ✔

b. use liposome ✔

c. get genes of choice into the vesicle/liposome ✔

d. get protoplast to fuse with the vesicle/liposome ✔

This question was not answered well by most candidates. Students struggled to explain concepts like how protoplasts are made, or the role of vesicles/liposomes.

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.

cooling- or heating-water systems / rocks at the bottom of a river / teeth «of most animals» / prepared on sewage treatment plants / boat hulls / medical catheters

Accept other verified examples

a. have «new» properties that are not present in the individual microorganisms 

b. organisms form a matrix «EPS» / biofilms have a complex architecture 

c. increased resistance to antibiotics/treatments
OR
bioluminescence 

d. biofilms can be formed by different types of micro organisms that interact/cooperate 

e. quorum sensing
OR
high population/cell density determines expression of genes

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.

Aims [1 mark]
a. the addition of organisms to remove environmental contaminants
OR
the addition of organisms to convert toxic compounds to non-toxic products ✔

Methods [1 max]
b. physical and chemical procedures can be combined with bioremediation ✔

c. e.g. clean-up of oil spills using bacteria
OR
clean-up of heavy metals using plants and incineration ✔

Accept other valid methods involving living organisms.
Do not accept physical methods alone.

to observe that it was stable
OR
to confirm that change was permanent
OR
to confirm that merB gene still present after many generations ✔

a. used to clean up oil
OR
used to clean up mercury ✔

b. used after other clean-up methods have been used ✔

c. nutrient concentrations have to be sufficient to support the maximal growth rate of the bacteria throughout the clean-up operation ✔

d. converts methylmercury into inorganic mercury «less toxic» ✔

e. converts oil into CO₂, water and simpler compounds «non-pollutant» ✔

f. produces extracellular digestive enzymes «lipase» ✔

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. organisms with similar gene sequence could be used as models for specific gene functions (in humans) 

b. it is possible for researchers to learn about the gene function faster/more easily as fewer genes (in model) 

c. can predict effects in other organisms 

d. some research may be unethical in humans

a. results from BLASTn or BLASTp /genomic or proteomic sequences can be used 

b. compared with data from available organisms 

c. matches based on the number of similarities in the sequence are identified 

d. similarities in sequence may be caused by evolution or by chance 

e. computer programs (use sequence alignments) to suggest evolutionary relationships / cladograms can be constructed

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.

a. mRNA is purified from «normal and cancer» cells 

b. only expressed/transcribed genes produce mRNA 

c. cDNA is «a complementary DNA copy» synthesized from mRNA «by using reverse transcriptase/transcription»

a. red spots are genes only expressed in cancer cells 

b. green spots are genes only expressed in normal cells 

c. yellow spots mean that genes are expressed in both normal and cancer cells 

d. red spots mean that this gene is missing/not active in normal cells
OR
those «red spot» genes could be promoting cancer growth 

e. green spots mean that this gene is missing in cancer cells
OR
missing genes could be an inhibitor of cancer

Explain how antithrombin can be produced by biopharming. 

a. biopharming is production of recombinant proteins/drugs by using transgenic animals ✔ 

b. the gene for antithrombin is cut from human DNA ✔

c. the gene is combined with the gene producing milk protein/casein ✔

d. the recombinant gene is inserted into an embryo of the goat ✔

e. the embryo is implanted in a female goat ✔

f. a promoter sequence is transferred with the gene for antithrombin to ensure that the gene is activated in cells that produce milk ✔

g. a signal sequence is transferred with the gene ✔

h. to ensure that the mRNA is translated by ER ribosomes ✔

i. the offspring of the goat are tested for antithrombin in their milk ✔

j. offspring with the recombinant gene are selected for breeding ✔

k. antithrombin is isolated from the milk and purified ✔

l. many goats with the recombinant gene allow large scale production of antithrombin ✔

Accept other milk producing farm animal eg: goat, sheep, camel

Although many referred to the specific example of the gene for antithrombin being inserted into a goat embryo, students seemed to find it harder to define biopharming or outline specific steps needed, such as the promoter sequence being transferred with the gene for antithrombin to ensure that the gene is activated in cells that produce milk.

Discuss biopharming.

a. produces useful pharmaceuticals/drugs/proteins

b. inserts genetic material/genes into host plants/animals

c. produces more complex drugs/proteins than prokaryotic organisms

    OR

    no post-translation modification with prokaryotes «so no complex proteins»

d. valid example

Allow verifiable examples, eg: antithrombin/coagulation factors «in goats», development of Norwalk virus/
cholera toxin vaccines «in tomatoes»

e. issues regarding contamination of other organisms

    OR

    possible ecological effects

f. plants process proteins differently than humans

g. proteins produced by plants may cause allergic reaction

h. some proteins are intellectual property

i. example of ethical issue

[Max 6 Marks]

Discuss the use of microorganisms in bioremediation.

a. pollutants metabolized by microorganisms 

b. microorganisms useful in bioremediation because they multiply very quickly «by binary fission» 

c. are varied in their metabolism/inorganic reactions 

d. use pollutants as energy/carbon sources 

e. use pollutants as electron acceptors «in cellular respiration» 

f. bioremediation may convert heavy metals into less toxic forms «in the food chain» 

g. pollution incidents can involve bioremediation combined with physical/chemical procedures 

h. preferable to physical methods because of cost/time 

i. preferable to chemical methods which can leave toxic residues 

j. different microorganisms to be used dependent on abiotic conditions 

k. one named organism

Discuss the use of microarrays in the diagnosis of disease.

a. DNA spots/probes/sequences attached to solid surface/microarray ✔

b. mRNA from healthy tissue/cell isolated and converted to cDNA
OR
mRNA from cancer tissue/cell isolated and converted to cDNA ✔

c. conversion to cDNA by reverse transcriptase ✔

d. fluorescent dye linked to copy DNA/cDNA ✔

e. cancer cDNA colored with a different dye from the healthy cDNA ✔ Accept named colour.

f. cDNA binds to/hybridizes with probes that have complementary base sequences ✔

g. microarray rinsed to remove cDNA that has not hybridized ✔

h. microarray exposed to laser light which causes fluorescent dye to give off light ✔

i. fluorescence shows which probes have hybridized
OR
fluorescence shows which sequences were in the tissue/sample ✔

j. hybridized probe shows gene expression
OR
hybridized probe helps in diagnosis of disease ✔

k. infection by pathogen can be detected by presence of its genetic material ✔

Most candidates had some knowledge of microarrays and could outline the outcome of their usage; some answers nevertheless were not displayed in a very logical order, missing the details of hybridization. Many specified only DNA where cDNA should have been mentioned.

Explain the formation of biofilms and the problems associated with their formation.

Formation
a. biofilm is a group of microorganisms embedded in a «exopolysaccharide/EPS» matrix

b. microorganisms adhere on a surface/to each other

c. cells are able to communicate/cooperate via quorum sensing
OR
secrete molecules that facilitate the aggregate adhering to the surface
OR
facilitate individual cells sticking together/OWTTE

d. phenotypic shift in behaviour
OR
emergent properties appear
OR
differential regulation of genes

Problems
e. «formation of biofilms» in the body facilitates infections/OWTTE 

Accept any verifiable health problem caused by biofilms, e.g. dental plaque causing caries, lung infection in cystic fibrosis patients, etc.

f. clogging/corrosion of pipes in water systems

g. transfer of microorganisms in ballast water

h. contamination of surfaces in food production

i. highly resistant to antimicrobial agents/antibiotics

j. EPS provides a physical barrier to the entry of the antibiotic «into the colony»

Award [5 max] if only problems are mentioned.