Genetic Engineering and Biotech

What is artificial selection?

Selective breeding of a species by humans resulting in a change in allele frequency.

What is inbreeding?

Breeding of closely related individuals

Describe a problem with inbreeding.

Reducing the gene pool and hence genetic diversity --> reduces their chance to evolve and adapt to environmental changes / Higher chance of inheriting recessive alleles that may cause genetic disorders

What is the effect of artificial selection on genetic diversity?

Decrease

What is a gene bank?

Storage of genomes within organisms so as to provide possible new alleles for future artificial selection.

What are the 2 main ethical objections to artificial selection?

Loss of a species' natural characteristics / behaviour, inbreeding depression

What is a DNA probe?

Single stranded piece of DNA that is complementary to a gene of interest. It is attached to a marker (tag) to identify the presence of specific genes 

Which part(s) of the genome are compared in DNA profiling for forensics

Non-coding regions (introns, STRs, VNTRs, minisatellites, microsatellites)

Put these steps for creating a DNA profile in the correct order: Amplification, Visualisation, Extraction, Digestion, Separation

Extraction, Amplification, Digestion, Separation, Visualisation

Give 2 uses of DNA profiles.

Paternity testing, forensic investigations, determining how closely related organisms/species are, genetic screening for disease risk

State three potential sources of DNA at a crime scene

Hair, blood, skin cells, semen, saliva etc. 

Two primers are needed in PCR. What is a primer?

Short, single-stranded DNA fragment (used to 'tell' the DNA polymerase the part of DNA to copy)

The first step of a PCR cycle is to heat to 95oC. What happens in this step?

Double stranded DNA is searated into two single strands (hydrogen bonds break)

What is special about Taq polymerase compared to other DNA polymerases?

Very stable at high temperatures so does not denature

The second stage of PCR (after heating to 95oC) is to cool to around 55oC. What is the purpose of this step?

Allow primers to anneal (bind)

A PCR mixture must contain DNA polymerase, free nucleotides, buffer, primers and what?

The target DNA sequence to be amplified

If the number of DNA fragments doubles in each PCR cycle, how many fragments will there be if one piece of DNA goes through 15 cycles of PCR?

32768

What does PCR stand for?

Polymerase chain reaction

In gel electrophoresis, is DNA attracted to the anode (+ve) or cathode (-ve)

Anode (DNA is negatively charged)

Decribe the movement of small fragments compared to larger fragments in gel electrophoresis

Smaller fragments move further/faster

In gel electrophoresis, what are DNA markers?

Mixtures of DNA molecules of known size. They are run in one lane and are used to estimate the sizes of the other DNA samples

In gel electrophoresis, what is the gel made of?

Agarose

What is the purpose of gel electrophoresis?

Separate DNA fragments in order of size

What is a genome?

genes or genetic material present in a cell or organism

Give two reasons why new DNA sequencing techniques have improved upon old methods

Quicker, cheaper

In order to use the Sanger sequencing method, apart from the DNA sample to be sequenced, what are the four other chemicals needed?

Taq DNA polymerase, a primer, normal nucleotides, chain terminator nucleotides (ddNTPs)

How is a terminator base different from a normal nucleotide?

Has a hydrogen instead of hydroxyl group on C3 of the deoxyribose sugar

How are the terminator bases altered in order for the sequence of nucleotide to be seen or detected?

Has a coloured fluorescent tag - 4 different colours for different bases

Why would the addition of the terminator base stop further extension of the DNA strand in DNA sequencing?

Terminator bases do not have hydroxyl group on C3 of deoxyribose, therefore cannot form phosphodiester bonds with the next nucleotide

What is the first step in sequencing a whole genome?

Cut the genome into smaller fragments (and clone into BACs to make a clone library)

How are the DNA fragments separated and read in DNA sequencing?

Gel electrophoresis in minute capillary tubes --> separate DNA fragments by size --> They can be read when passing through a laser that reads the colour as they pass through

Briefly describe how next-generation sequencing works.

Automated, high-throughput sequencing process: millions of DNA fragments are attached onto a surface and sequenced as clusters at the same time

Explpain how genome sequencing can help identify evolutionary relationships.

Closer % match of genome sequence means less time since the two speices diverged from a common ancestor. 

Bioinformatics is transforming epidemiology. What is epidemiology?

The incidence, distribution, and possible control of diseases (and other factors relating to health)

What’s the difference between Bioinformatics and Computational Biology?

Bioinformatics – development of software to process large amount of data produced from sequencing etc. Computational Biology – using that data to create computer models and test theories

Name one benefit to sequencing pathogens' genomes.

Find out the source of an infection / Identify antibiotic-resistant bacteria strains to evaluate the use of antibiotics / monitor a disease outbreak / find useful targets in genome when developing new drugs

What is proteomics?

Study and amino acid sequencing of an organism's entire protein complement

The amino acid sequence is not always what would be predicted from the genome sequence itself. Suggest two reasons why.

1.) Genomes have exons and introns --> introns are removed + spliceosomes join different exons together in different ways to make different proteins; 2.) Protein modification by Golgi apparatus

What is DNA barcoding?

Identifying particular DNA sections that are common to all species but vary between them --> useful as comparison for evidence for evolution

Once scientists have sequenced a gene they can work out the amino acid sequence of the protein it codes for. How? 

Triplet code has been worked out (i.e. all 64 combinations of three bases are known and decoded)

What is synthetic biology?

Creating biological molecules from scratch (e.g. "printing" a sequence of DNA)

What is recombinant DNA?

DNA molecule artificially generated from different origins (often different species)

What is a restriction endonuclease?

Enzyme that cuts a double stranded DNA fragment at a specfic place (its restriction site)

What is meant when a restriction enzyme recognition site is said to be 'palindromic'?

It has the same sequence on both strands (reading from 5' to 3')

What are 'sticky ends' in genetic engineering?

Complementary single stranded 'overhangs' of DNA which can be used to stick two DNA fragments together.

Do restriction enzymes catalyse condensation or hydrolysis reactions?

Hydrolysis

What is another method of getting the desired gene apart from using restriction endonucleases?

Use reverse transcriptases

What is a reverse transcriptase?

Enzyme that makes a complementary DNA (cDNA) from the isolated mRNA made from the desired gene

What does it mean when we say that two DNA fragments are 'annealed'?

Joined together (compatable sticky ends)

What process could you describe as the reverse of restriction digestion?

DNA Ligation

Name a common vector used in genetic engineering.

Plasmids (also cosmids, viruses, artificial chromosomes such as BACS, liposomes) 

What is the role of DNA Ligase?

Joins DNA backbone/sugar phosphate backbone (makes phosphodiester bonds) of two DNA fragments together

How are restriction enzymes used in genetic modification?

Cut plasmid, isolate gene, producing sticky ends

What is electroporation?

Using an electrical current to make cell membranes more porous (to allow plasmids to enter)

What is electofusion?

Pass tiny electric currents to the membranes of two different cells to fuse them together, forming a hybrid/polyploid cell

What is a transgenic organism?

One that has been genetically altered to include genetic material from another organism

Suggest a reason for genetically modifying a plant.

Insect resistance, drought tolerance, pesticide resistance, faster growth, better flavour, slower ripening etc.

State a negative aspect of genetically modifying plants.

Create monocultures (susceptible to extinction), chance of gene transfer to create superweeds, expensive to buy

What is 'pharming'?

Making medicinal drugs (pharmaceuticals) from genetically modified organisms

Give an ethical positive and negative to “pharming”.

Positive– easier/cheaper production of medicine. Negative – long term effects to health of organism unknown, patenting issues.

Give a positive and negative ethical issue of geneticially engineering pathogens

Positive - may be able to engineer them to attack cancer cells. Negaive - Risk of mutation/revertion and therefore cause major outbreak of disease, intentialal biowarfare

What is gene therapy?

Altering alleles to treat genetic diseases

What’s the difference between somatic cell therapy and germ line cell therapy?

Somatic - altering genes in body cells. Germ Line – altering genes in gametes or zygote

State a negative feature of somatic cell gene therapy.

Effects are often short-lived, multipple treatments may be needed, hard to target some body cells, allele could go to wrong place and cause a problem, expensive, where do we draw the line? (e.g. should we 'fix' shortsightedness, baldness, hair colour etc.)

State a negative feature of germline gene therapy.

Offspring will also carry altered genes - may be unknown long term effects. 

Why is it harder to treat genetic disorders caused by dominant alleles than disorders caused by recessive alleles?

Recessive allele treatment just needs addition of the "correct" allele anywhere in genome. Treatment of a dominant condition requires that specific gene to be disrupted/silenced. This requires more sppecific placement of inserted DNA.

State whether each type of gene therepy (somatic and germline) is legal or illegal.

Somatic = legal.   Germline = illegal.

State 3 ways in which plants clone themselves.

Sending out runners, making suckers, producing bulbs, producing corms, producing immature plants on the leaves (e.g. kalanchoe),  producing tubers, 

Define micropropagation

Growing large numbers of plants from meristem tissue taken from a sample plant

Define tissue culture.

Growing new tissues, organs or plants from certain tissues cut from sample plants

In what circumstances would micropropagation be used?

If the plant doesn't produce many seeds, doesn't respond well to natural cloning, is rare, needs to be pathogen free, is GM or selectively bred

Outline the steps involved in micropropagation.

Cells removed from the shoot > cells /explants are sterilied before being placed onto the sterile nutrient medium > explants divide to form a callus, small clumps of undifferentiated cells > callus transferred to a new agar medium > plantlets transferred to compost

State 3 advantages of cloning plants.

Can produce lots of plants quickly, if aseptic technique is followed the new plants will be disease free, plants can be chosen with desirable traits (high-yielding, pest-resistant, disease-resistant, frost-resistant), infertile plants can be grown, harvesting is easier as all plants have the same genotype, 

State 3 disadvantages of cloning plants. 

Expensive, can fail due to microbial contamination, all cloned offspring are susceptible to the same pest or disease (monoculture), reduces genetic variation in a species.

Describe the difference between micropropagation and tissue culture.

Micropropagation produces a large number of plants from a small sample of plant material whereas tissue culture is growing plant cells in an artificial medium, forming large numbers of plantlets (i.e. the first step in micropropagation).

Describe how to take cuttings.

Cut a 10cm section from a non-flowering stem of the plant > remove the top leaves > dip the cut end into a rooting powder > push the plant into the compost > add water to the compost and cover the plant with a plastic bag

What are explants?

Small pieces of plants cuttings taken

How are explants processed before cultured for cloning, and why?

Sterilised using bleach/ethanol/sodium dichloroisocyanurate --> avoid growth of pathogens or microorganisms that may compete with explants for resources during growth (aseptic reasons)

What chemicals need to be added to the nutrient agar plates to induce plant development during micropropagation? Give two examples.

Plant hormones: Auxins for shoot growth, cytokinins for root growth

State the term given to a ball of unspecialised plant cells produced during micropropagation.

Callus

What do we call natural human clones?

Twins.

Outline how twins / natural animal clones are formed.

One sperm fertilises one egg > mitosis produces a ball of cells called an early enbryo > the embryo splits and implants in the uterus lining where mitosis continues.

Outline how embryo twinning works.

One sperm fertilises one egg > zygote divides to form an embryo > the embryo is split into separate cells > the cells divide by mitosis to form genetically identical embryos > each embryo is planted into a surrogate > offspring are born which are genetically identical to each other.

In artificial twinning, explain why the cow needs to be treated with hormones as the first step.

So it super-ovulates to release mature eggs (for collection)

In artificial twinning,the offspring are genetically identical to whom?

To each other (all offpsring are clones of each other)

Which method is a type of reproductive cloning - Artificial twinning or somatic cell nuclear transfer?

Somatic cell nuclear transfer (SCNT)

Outline how somatic cell nuclear transfer cloning works.

A somatic cell is obtained and the nucleus is removed > a donor egg is obtained and enucleated > the somatic cell nucleus is inserted into the enucleated oocyte > electrofusion of the host cell and new nucleus > the transformed egg divides in vitro > the embryo is transferred into a surrogate uterus > the clone is born.

In SCNT, which cell becomes enucleated?

Mature egg cell/ovum from a female animal

How is the enucleated egg cell fused with the somatic cell nucleus?

Electrofusion

Explain why the offspring in SCNT is not an exact clone of the nucleus donor.

Different mitochondrial DNA as mitochondria are inherited from the egg cell donor

State 3 advantages of cloning animals.

Desirable traits are selected for and guaranteed to be passed on, infertile animals can be reproduced, do not need to wait for breeding season, increase populations of endangered species, 

State 3 disadvantages of cloning animals. 

Difficult time-consuming and expensive, all are susceptible to the same disease, undesirable characteristics also always passed on, clones tend not to live as long as natural offspring

Give one use of animal cloning.

Farming / Pharming / Restore endangered animal populations

Name the microorganism used in baking.

Yeast (often Saccharomyces sp.)

Explain why bread rises.

Yeast respires, releasing carbon dioxide which gets trapped between crosslinked gluten molecules.  As the temperature rises, the carbon dioxide bubbles expand.

Name the microorganism used in brewing.

Yeast (often Saccharomyces sp.)

Name the type of respiration used by microorganisms during brewing and state the products  of this.

Anaerobic respiration (fermentation), producing carbon dioxide and ethanol.

Give the balanced symbol equation for fermentation of glucose.

C6H12O6 --> 2C2H5OH + 2CO2

Name the type of organism used in making cheese.

Bacteria (e.g. Lactococci and Lactobacilli sp)

Which enzyme is used in cheese making and what is the source of this? 

Chymosin (from rennet), from the stomach of a calf

Describe the steps involved in making vegetarian chymosin (by genetic engineering).

Use genetic engineering - isolate the gene for chymosin from a cow > cut the gene using restriction endonucleases > remove a plasmid from a prokaryotic cell > cut the plasmid using the same restricton enzyme > insert the gene into the plasmid, with H bonds holding the complementary base pairs in place > use DNA ligase to form phosphodiester bonds between the gene and the plasmid > insert the transformed plasmid into a prokaryotic cell > provide aseptic conditions with plentiful nutrients > the prokaryote will express the gene as it divides by binary fission.

Name the type of organism used in making yogurt.

Bacteria (e.g. Lactobacillus or Streptococcus)

Name the type of organism involved in producing penicillin.

Fungus (Penicillium sp.)

Which microorganism is used to make single cell protein / mycoprotein?

Fungus (Fusarium sp.)

State the reactants and conditions in the fermenter when mycoprotein is made and explain why each is needed.

Fusrium fungus - to produce the mycoprotein; glucose - respiratory substrate; ammonia - to provide a nitrogen source; sterile oxygen - to ensure aerobic respiration without contaminating the mixture; pH and temperature at an optimum - to ensure maximum growth; water cooling jacket - to remove thermal energy released in respiration; stirring paddles - to ensure thorough mixing of reactants;   

State 3 advantages of producing and consuming SCP.

Suitable for vegetarians; high protein, low fat; lots can be produced in a short space of time; does not require a lot of land as fermenters are built vertically; can be transformed into different flavours and textures

State 3 disadvantages of producing and consuming SCP.

Risk of contamination if fermenter/ reactants are not sterile; have to extract, purify and flavour the mycoprotein; some people may not want to eat fungal protein

Is penicillin produced in batch fermentation or continuous fermentation?

Batch

Where did insulin historically come from?

Animal pancreases (e.g. pigs)

What was the problem with using insulin from animals?

Not very effective as not the same structure to human insulin, difficult to extract in large quantities, ethical concerns about the use of animals to provide insulin, not suitable for use by people with particular beliefs

Describe the steps involved in making synthetic insulin.

Use genetic engineering - isolate the gene for insulin from a human pancreas > cut the gene using restriction endonucleases > remove a plasmid from a prokaryotic cell > cut the plasmid using the same restricton enzyme > insert the gene into the plasmid, with H bonds holding the complementary base pairs in place > use DNA ligase to form phosphodiester bonds between the gene and the plasmid > insert the transformed plasmid into a prokaryotic cell > provide aseptic conditions with plentiful nutrients > the prokaryote will express the gene as it divides by binary fission.

Is insulin produced in batch fermentation or continuous fermentation?

Continuous

Define bioremediation.

The use of microorganisms to clean the soil and underground water on polluted sites.

Describe how bioremediation works.

Microorganisms covert toxic substances to less harmful substances.

What is the difference between in situ and ex situ bioremediation?

Contaminants are broken down on-site during in situ and taken elsewhere during ex situ.

Why is a source of carbon needed when growing microorganisms?

To provide a respiratory substrate

Why is a source of nitrogen needed when growing microorganisms?

To allow protein synthesis to occur

What is the jelly-like substance called on which microorganisms are often grown?

Agar

What is meant by a closed culture?

A culture which has no exchange of nutrients or gases with the external environment.

Sketch and label the growth curve for a population of microorganisms in a closed culture.

Correct curve sketched, time x axis, population size y axis, lag phase, exponential phase, stationary phase, death/ decline phase all labelled

Describe the lag phase of the bacterial growth curve.

The population does not grow quickly.  Reproduction rate = death rate.

Explain the lag phase of the bacterial growth curve.

Population is small and is adjusting to new conditions - taking up water / cell growth / synthesising proteins

Describe the log / exponential phase of the bacterial growth curve.

The population grows quickly.  Reproduction rate > death rate.

Explain the log / exponential phase of the bacterial growth curve.

Population has adjusted; microorganisms have enzymes they need; sufficient space and nutrients

Describe the stationary phase of the bacterial growth curve.

The population becomes static / no population growth.  Reproduction rate = death rate

Explain the stationary phase of the bacterial growth curve.

Nutrients and space are running out; waste is accumulating

Describe the death / decline phase of the bacterial growth curve.

Population begins to fall. Reproduction rate < death rate.

Explain the death / decline phase of the bacterial growth curve.

Nutrients run out; concentration of waste products becomes toxic.

Give 1 / 2 / 3 steps you'd take to work aseptically.

Wash your hands, disinfect the working area, work near a Bunsen burner, flame the neck of any bottles upon opening and closing, only open the lid of the Petri dish enough to inoculate the plate, flame any glassware or metal equipment before use

Explain why you'd work near a Bunsen burner when working aseptically.

The air warms and rises, preventing any air-borne microorganisms from settling / creates an area of sterile air in which the microbiologist can work

How is the nutrient agar medium sterilised when preparing to grow microorganisms?

Heating in an autoclave

How is the equipment sterilised when preparing to grow microorganisms?

Heating in an autoclave

Describe the conditions when something is placed in an autoclave.

121oC, 15 minutes

Explain the conditions in an autoclave.

All living organisms are killed, including bacterial or fungal spores.

What is meant by inoculation?

The deliberate introduction of microorganisms to a sterile medium.

Name 1 / 2 / 3 / 4 ways of inoculating a medium.

Streaking / seeding / spreading / using a sterile cotton swab to collect microorgansisms and wipe them over the medium

Describe the plate streaking technique.

A drop is transfered to the medium using an inoculating loop and drawn into a streak

Describe the plate seeding technique.

A sterile pipette transfers a small drop of liquid medium to the agar surface or to the Petri dish before the agar is poured

Describe the plate spreading technique.

A sterile glass spreader spreads the inoculating drop over the surface of the agar

How is the agar plate stored after inoculation?

Taped at 4 points, incubated at 25oC, placed upside down, 

Explain the reasons for not sealing the lid to the agar plate using sticky tape after inoculation.

Allows oxygen to enter - preventing the growth of anaerobic pathogens

Explain the reasons for incubating at 25oC after inoculation.

Incubating at 25oC prevents growth of pathogens

Explain the reasons for storing the agar plate upside down after inoculation.

This prevents drops of condensation falling onto the agar; also prevents the agar drying out too quickly

When looking at your plate, how would you distinguish bacterial colonies from fungi?

Bacterial colonies are shiny or smooth whereas fungi look like cotton wool with fluffy hyphae

Why would serial dilutions be used in microbiology?

To determine the population size and growth rate of a population of microorganisms

Describe how you'd make a serial dilution that has a dilution factor of 10.

Use 1cm3 of broth and 9cm3 of distilled water.

Once you have made your dilution series, describe what can be done with it.

Place 1 drop of each dilution onto a sterile agar plate.  Allow colonies to form.  Count the number of colonies on the plate which is easiest to count.  Then multiply by the dilution factor.

Describe the difference between primary and secondary metabolites.

Primary metabolites are produced during the normal activities of the microorganism during the log phase whereas secondary metabolites are produced during the stationary phase.

Batch or continuous culture: carried out in a closed fermenter, with nothing added or removed?

Batch

Batch or continuous culture: microorganisms are left for a set period of time?

Batch

Batch or continuous culture: carried out in an open fermenter, with nutrients added and products removed?

Continuous

Batch or continuous culture: no idle time and greater product yields?

Continuous

Give an advantage of batch culture over continuous culture.

The fermenter can be used for different reactions with each separate use

Give a disadvantage of batch culture over continuous culture.

There is lots of idle time between use therefore higher costs

Give a disadvantage of continuous culture over batch culture.

Higher risk of contamination due to constant additions and adjustments

How is penicillin produced - batch culture or continuous culture?

Batch (fermentation)

How are bioreactors cooled?

Using a water jacket

Why is it necessary to cool bioreactors?

Reactions are exothermic - heat generated can denature enzymes

Which gas is added to aerobic fermenters?

Oxygen

Describe and explain the condition of all reactants added to a fermenter.

Sterile - to avoid contamination of the product / to avoid competition from other microorganisms for reactants

How are substrates and organisms mixed in a fermenter?

Motor with stirrers / mixing blades (impellers)

How is the pH monitored in a fermenter?

Using an electronic pH probe

Why is it necessary to monitor and adjust the pH in bioreactors?

Enzyme activity (and therefore growth) is affected by extremes of pH

How are fermenters sterilised?

With superheated steam

Define immobolised enzyme

An enzyme that is held in place and not free to diffuse through the solution

Give 1 / 2 / 3 advantages of using immobilised enzymes.

Extraction costs are lower as enzymes do not mix with the product / the enzymes can be easily reused / a continuous process is made easier as there are no cells requiring nutrients and releasing waste products / the enzymes are protected from extreme conditions so don't get easily denatured.

Give 1 / 2 disadvantages of using immobilised enzymes.

Setting up the immobilised enzymes is more expensive / immobilised enzymes are less active so the reaction is slower

Name the method described: enzyme molecules are bound to a supporting surface by hydrophobic interactions and ionic links.

Adsorption

Name the method described: enzyme molecules are separated from the reaction mixture by a partially permeable membrane

Membrane separation

Name the method described: enzyme molecules are bonded to a supporting surface by strong covalent bonds

Covalent bonding

Name the method described: enzyme molecules are trapped in a matrix that does not allow free movement

Entrapment

What materials can be used as a supporting surface when using adsorption as a technique to immobilise enzymes?

Unreactive material - eg. Clay, porous carbon, glass beads, resins.

Give one disadvantage of using adsorption as a method of immobilising enzymes.

Active site may be distorted so enzyme activity may reduce / enzymes can become detached and leak into reaction mixture so need separating or replacing

Give one disadvantage of using covalent bonding as a method of immobilising enzymes.

Can be expensive / can distort the active site

Give one disadvantage of using entrapment as a method of immobilising enzymes.

Substrate needs to diffuse in to the matrix / product needs to diffuse out so only suitable for processes where substrate and product are small

Why is it an advantage to convert glucose to fructose?

Used to produce high fructose corn syrup - much sweeter than sucrose

What is the role of lactase?

Converts lactose to glucose and galactose to produce lactose-free milk