HL - IB Biology Option B: Biotechnology and Bioinformatics

Production of biogas

- Bacteria first convert the organic materials to organic acids and alcohol. - Other bacteria convert these products into acetate (CH₃COOH), CO₂ and H₂ gas. - Methanogens then create methane either by: - breaking down the acetate or CH₃COOH → CH₄ + CO₂ - combining CO₂ and H₂ CO₂ + 4H₂ → CH₄ + 2H₂O

Negative impacts of Biofilms

- Can form on plant surfaces and cause crop disease - Can cause diseases when present internally in animals - Can form on man-made structures resulting in long-term corrosion. - Adhesion to ship hulls can cause deterioration of pipes etc. (aka biofouling).

4 reasons biofilms are so effective

- Cooperate with one another - Develop quickly - Hold themselves to each other and to the surface strongly using extracellular polymeric substances (EPS) - Can become resistant to various antimicrobial agents

Positive impacts of Biofilms

- Form an important part of aquatic food chains and also filter sediments to maintain water quality. - Are actively involved in decomposition of biotic debris and nutrient recycling. - Form symbiotic relationships with plants - Constructive purposed such as bioremediation and sewage treatment

Genetically modified crops can also help consumers by...

- Improving the nutritional value of the crop (eg. golden rice produces more β-carotene) - Enabling certain crops to produce toxins that can kill insects and thus reduce the need for insecticides (eg. BT corn is toxic to the corn borer) - Enabling crops to behave as edible vaccines by possessing antigenic fragments of specific pathogens (eg. GM bananas could vaccinate against Hepatitis-B) All of these are examples of 'novel products' as they are things that the plant could not produce before.

Glyphosate resistance in Soybeans Pt. III: Advantages

- Reduced need of ploughing therefore reduced loss of topsoil (tilage) - Reduced fossil fuel usage - Weed regrowth rarely occurs i.e. better weed management - Glyphosate is rapidly degraded by soil microbes ∴ limited soil contamination - Glyphosate has very low level toxicity in mammals and is not retained much in insects.

How does a microarray work?

- Reverse transcriptase is used to make a DNA copy (cDNA) of the base sequence of each type of mRNA in a tissue. - Fluorescent dye is linked to each cDNA. - The microarray is exposed to the cDNAs long enough for hybridisation between fixed probes and cDNAs that have complementary base sequences. - The microarray is the. rinsed to remove the cDNA that has not hybridised. - It is then exposed to laser light which causes the fluorescent dye to emit light. This shows which probes on the microarray have hybridised with cDNA and thus which mRNA sequences there were in the tissue.

Genetic engineering is carried out by the following steps:

- The gene of interest must be identified and isolated (DNA extraction). - The gene must be delivered into the cells of the appropriate host organism (transgenic cells). - The transgenic cells must be harvested for successful gene expression.

Glyphosate resistance in Soybeans Pt. IV: Disadvantages

- These crops could become weeds themselves thus requiring tillage - Could potentially reduce biodiversity by eliminating weeds as food sources in food chains - Unintended effects like allergies - Effects of sub-lethal doses of glyphosate in plants and animals has not been well studied - Resistance could spread to non-target plants, i.e. crops-contamination, which would reduce the effectiveness of herbicides

3 Important properties of microorganisms

- are metabolically diverse - are small, so can be cultivated in large numbers - grow rapidly

Biofilm attachment

- attachment to a surface through weak reversible associations - over time, they form more permanent, irreversible attachments using adhesion structures - form a monolayer which grows and divides - produces an exopolymer matrix to encapsulate growing colony i.e unattached cells → surface growth → monolayer → microcolony (blob) → biofilm (+exopolymer matrix)

Optimum conditions and how they are maintained inside a fermenter:

- pH and temperature are monitored using probes. - cooling jacket with cool water flowing through surrounds the vessel to combat heat that builds up as metabolic waste. - sedimentation of microbes is prevented by an impeller which is a rotating set of paddles. - sterile air is bubbles through for aerobic reactions. - pressure gauge detects gas build-up and allows waste gases to escape.

Gram positive vs gram negative bacteria

- plasma membrane of phospholipids and proteins (+ve and -ve) - a thick layer of peptidoglycan (+ve) - a thin layer of peptidoglycan (-ve) - outer membrane of lipopolysaccharide and protein (-ve only)

How ELISA is carried out

1. Capture molecules are linked to the surface of a well on a plastic microtitre plate. The plate has many small wells - each is used for one test. 2. The sample is exposed to the surface to let any target molecule bind to a capture molecule. 3. Antibodies that have been linked to an enzyme are added. These antibodies bind to any target molecules that are not bound to absorbed target molecules. 4. The surface is rinsed to remove all the enzymes that are not bound to absorbed target molecules. 5. A substrate is added so that the enzyme changes to a different colour, indicated the presence of a target molecule.

Gene delivery systems

1. Chemical methods (Calcium chloride, liposomes) 2. Physical delivery (electroporation, microinjection, biolistics) 3. Vector delivery genes (A. tumefaciens, TMV)

Two types of marker genes:

1. Screening markers 2. Selectable markers (aka antibiotic resistance markers or ARM's)

How is gram staining carried out?

1. Smear a small sample of pure bacterial culture on a micro slide with an inoculating loop. 2. Pass through flame to fix bacteria to the slide. 3. Stain with crystal violet for 30 seconds. 4. Treat with grams iodine for 30 seconds - to bind crystal violet to the outer surface of the bacteria. 5. Decolorize with alcohol for 30 seconds - to dissolve the outer membrane of gram-negative bacteria and remove the crystal violet staining. 6. Counterstain with safranin (red) for 30 seconds, then rinse and blot dry.

Open Reading Frames (ORF)

A continuous stretch of DNA that contains a start codon, a stop codon and a minimum of a 100 codons (i.e. 300 nucleotides). The start codon MUST be on the 5' end of the DNA sense strand (therefore will also be on the 5' end of the mRNA).

Biofilms

A cooperative aggregate of microorganisms via bacterial adherence associated with a surface and enclosed in an extra-cellular matrix of adhesive polysaccharides also called extra-cellular polymeric substances (EPS).

Marker genes

A gene that is linked to sequences of target DNA. It is used by researchers to identify if recombinant DNA has been taken up into cells.

Amflora potatoes Pt. II: The solution

A gene with the same base sequence as GBSS, but reversed was inserted into the potato cells. The mRNA transcribed from this gene has a base sequence that is complementary to the mRNA transcribed from the normal GBSS gene, i.e. it has the sequence for the ANTISENSE strand. ∴ the ANTISENSE mRNA binds with the SENSE mRNA to form double-stranded RNA. Thus, this cannot be translated by ribosomes and GBSS cannot be produced, so more than 99% of starch produced by 'Amflora' potatoes is amylopectin.

What is a microarray?

A microarray is a small surface with a large range of probe sequences adhering to it. They test for specific mRNA sequences in tissues.

BLASTn

A nucleotide sequence alignment tool. Using this program, a DNA sequence can be input and the software will return the most similar DNA sequences from the DNA database specified by the user.

EPS is composed of...

A polymeric conglomeration of: - extracellular polysaccharides - proteins - lipids - DNA

BLASTp

A protein alignment tool. BLASTp allows the user to query a protein sequence and get the most similar protein sequence from the specified database.

Fermenters

A sterile, enclosed vessel that maintains optimal conditions for microorganic growth. They undergo fermentation to produce large quantities of a desired metabolite for commercial use.

Gram-negative bacteria

After gram staining, they will turn RED/ PINK.

Gram-positive bacteria

After gram staining, they will turn VIOLET.

Databases

Allow for easy access to information that can be easily accessible from all around the world.

Transgenic organisms

An organism is transgenic if it has been genetically modified with a gene from another organism - i.e. they produce proteins that were not previously part of their species' proteome. The new gene which is expressed is the target gene.

BLAST

Basic Local Alignment Search Tool It is an algorithm for comparing biological sequence information.

Example of problems posed by biofilms: Clogging and corrosion of sewage pipes

Biofilms can form inside pipes or other parts of water supply systems. Bacteria in the interior of these biofilms can be resistant to disinfectants. Bacteriophages (virus that can kill bacteria) are used to remove biofilms. Biofilms made of E. coli are removed by the bacteriophage T4.

Citric acid

Citrate is commonly used as a flavour enhancer, preservative for manufactured foods and as an antioxidant. It is produced as an intermediate product of the Krebs cycle under aerobic conditions; therefore it is a primary metabolite as it essential to the growth and development of the microorganism. It is produced from the cultures of the fungus Aspergillus niger.

Bioremediation for Oil spills (Crude Oil)

Crude oils biodegradable and hence oil spills are commonly dispersed by bacterial decomposition. Pseudomonas is a chemoheterotrophic species that can utilise crude oil as its primary carbon source and breaks it down to harmless products of H₂O and CO₂. Potassium and urea are needed to metabolise the oil at a faster rate, so these inorganic nutrients are sprayed onto the surface of the oil.

Detecting a predisposition to a genetic disease

Detected by the presence of genetic markers. These are particular alleles that either contribute to the disease or are genetically linked to genes that influence the disease. The marker is detected by microarrays or PCR and gel electrophoresis.

Bioremediation for Mercury contamination

Elemental mercury (metal form) is produced as an industrial pollutant and ends up in garbage dumps. It is converted to methyl mercury which is highly toxic by the bacteria Desulfovibrio desulfuricans. It builds up in food chains in aquatic ecosystems by biomagnification. Pseudomonas putida can convert methyl mercury to methane and mercury ions (soluble form) Other bacteria use this soluble ion as an electron acceptor resulting in insoluble elemental mercury being reformed.

How can gene function be studied?

Gene function can be studied using model organisms with similar sequences eg. Knockout technology in mice

Glyphosate resistance in Soybeans Pt. I: How A. tumefaciens is used

Glyphosate = weedkiller that kills all plants, so cannot be sprayed onto growing crops A strain of Agrobacterium tumefaciens has a tumour-inducing plasmid (Ti plasmid). A glyphosate resistance gene was inserted into the Ti plasmid along with a kanamycin resistance gene. These RECOMBINANT Ti plasmids were reinserted into the bacteria.

Production of Hepatitis B vaccine using TMV Pt. II: The development of the transgenic plant

Hepatitis B contains Hepatitis B small surface antigen (HBsAG). TMV has been genetically modified with the gene for making HBsAG from Hepatitis B. Tobacco plants are then infected with the transgenic TMV to produce HBsAG in bulk. The tobacco plants are harvested and dried, and when consumed, the HBsAG in it should stimulate the production of antibodies against Hepatitis B and therefore induce immunity to the disease.

Knockout technology in mice to determine gene function:

If a mouse gene has a high degree of similarity to a human gene, researchers can predict that the two genes carry out related functions. Therefore, by generating a knockout mouse without a gene of interest, these scientists may be able to determine the functions carried out by the related human gene.

Interpretation of ELISA test results

If colour change has occurred in a well, it means that the test in +ve and the target molecule is present in the sample. The stronger the colour, the more of the target molecule is present.

Analysis of microarrays

It involves examining the pattern of dots of the different possible colours. The pattern changes as gene expression changes. In tumour cells, marked changes in gene expression allow the detection and characterisation of cancer.

Biogas

It is a clean and renewable fuel source (i.e. combustible) produced by the anaerobic breakdown of organic matter by particular microorganisms. It is composed of CO₂, methane and water vapour. It can be produced from: - manure - sewage - agricultural waste - food scraps

Example: Deep-tank batch fermentation for the mass production of Penicillin.

It is a secondary metabolite as it is not directly involved in the growth and development of the microorganism. Optimum conditions: slightly alkaline pH at 24˚C Penicillin mould that produces antibiotic penicillin is grown in large industrial deep-batch fermenters of high capacity, by adding sugars and other key ingredients. Penicillin is separated from the mould typically after 6-8 days. Penicillin is purified to increase its antibiotic potential so it can be used as an antibacterial medicine.

Pathway Engineering

Modification/ manipulation of genetic/ regulatory processes of metabolic reactions to improve yields of metabolites of interest. This can include extending the range of substrates, removing by-products that slow the process down, and extending of the range of products (higher yield).

Bioremediation for Benzene

Offshore oil wells generate large volumes of saline wastewater contaminated with hydrocarbons. Benzene is a particular concern as it remains in the environment and is carcinogenic. Marinobacter hydrocarbonoclasticus is a halophilic (can live in highly saline conditions) archaen that degrades benzene.

Small-scale biogas production in fermenters

Optimum conditions: anaerobic, neutral pH (~7), constant temp. ≈ 35˚C Manure → Eubacteria (convert organic matter to CH₃COOH + CO₂ + H₂O) → Archeans/ methanogens (convert CH₃COOH + CO₂ + H₂O → CH₄ (g)) → Biofuel (methane) Biogas fermenters are airtight chambers with an inlet and vessel for biogas collection. Biogas forms at the top of the container and digested slurry is also produced which can be used as fertilizer. Fermenters can be linked to toilets and are typically used as a supplementary fuel source in rural communities.

Example: Continuous fermenter system for the mass production of Citric acid (Citrate).

Optimum conditions: high [O₂] and [sugar], low pH at 30˚C Carbohydrates are continuously added to maintain production → Fe²⁺ ions are excluded to prevent the progression go the Krebs cycle. Accumulated citric acid is continuously extracted.

Amflora potatoes Pt. I: The problem

Potatoes contain two kinds of starch: - 80% of amylopectin (branched form) - 20% of amylose (unbranched form) The amylopectin form of potato starch is used as an adhesive and a coating for paper. Granule-bound starch synthase (GBSS) is an enzyme used to make amylose but not amylopectin.

Emergent properties

Properties that a complex system possess, but not its individual members. Increased resistance to antimicrobial agents in an example of an emergent property in biofilms.

2. Selectable markers/ ARM's

Protects cells of organisms that have taken up new DNA from substances that would otherwise kill them. ARM's are often used to kill chloroplasts during the genetic modification of plant cells. Thus, any plant cells that haven't taken up the new DNA do are not resistant so can be targeted with suitable antibiotics that will destroy them.

Start and stop codons on mRNA

Start codon: AUG Stop codon: UAA, UGA, UAG

How to construct phylogenetic tree:

Step 1: Select a gene or protein common to a range of selected organisms. Examples of molecules which are ubiquitously found in many animals include haemoglobin and cytochrome c. Step 2: Copy the molecular sequence (DNA or amino acid) for each of the selected organisms - Use the GenBank database to identify relevant DNA or amino acid sequences - Sequences can be collated in a Word document and then saved as a document in plain text format (.txt) - Before each sequence, designate a species name preceded by a forward arrow (e.g. '>Human' or '>Chimpanzee') Step 3: Run a multiple alignment and generate a phylogeny tree (cladogram) - Use Clustal Omega align multiple DNA or amino acid sequences for comparison - Clustal Omega can generate branched phylograms after a sequence alignment is completed (select 'Phylogenetic Tree')

Phylogenetics

The analysis of evolutionary, or ancestral, relationships between taxa. Multiple sequence alignment is used in the study of phylogenetics.

How is knockout technology carried out?

The genetic information of individual cells is altered in some way creating a transgenic animal with an altered gene. Once this has been accomplished, the goal is to get embryonic stem cells transformed and inserted into early embryos. This results in animals with a genetic change in their germ-line cells that can pass the genetic alteration on to their offspring.

Tracking experiments

The movement of specific proteins in the body can be followed using tracking experiments in which a probe is attached to the protein. Radioactive probes can be located using a PET scan and fluorescent dyes used as probes are located with microscopes.

Quorum sensing

The regulation of gene expression in response to fluctuations in cell-population density. The ability of bacteria to communicate with one another. First few microorganisms produce inducers (signalling molecule) to do something. Surrounding microorganisms receive inducers through receptors, then makes more inducers for others.

Bioremediation

The use of microorganisms to detoxify and restore polluted and degraded ecosystems - their metabolic diversity is a plus

Detecting infectious diseases

They are caused by pathogens. Are detected by the presence of a pathogens genetic material, for example, with microarrays or PCR. An ELISA test can be used to detect a chemical produced by a pathogen that acts as an antigen.

1. Screening markers

They identify cells that have taken up new genes by their appearance: - a green fluorescent protein (GFP) makes cells glow under UV light - GUS assay which stains cells blue but it can also kill the cells - the blue-white method is used for both, bacterial and plant cells. It involves adding a bacterial gene coding for β-galactisidase enzyme which turns blue in the presence of the recombinant genes.

ELISA

This test can be used to detect antigens specific to a pathogen or antibodies indicating infection with the pathogen. It involves absorption of antibodies to antigens or vice versa and also a colour change caused by an enzyme. If antigens are the target molecule, then antibodies that bind to the antigen are used as the capture molecule.

Glyphosate resistance in Soybeans Pt. II: How resistance is developed.

This transgenic bacteria is now exposed to parts of the leaf of the soybean plant. The bacterium injects the Ti plasmid through a pious into plant cells and its DNA becomes incorporated into the chromosomes in the nucleus of the plant cell. Kanamycin is used to then kill leaf cells that have not taken up the Ti plasmid and a glyphosate-resistant variety of soybeans is developed from surviving cells.

Production of Hepatitis B vaccine using TMV Pt. I: About TMV

Tobacco mosaic virus (TMV) enters tobacco cells and uses the metabolism of the cell to translate some of its genes into proteins and to replicate its genetic material (RNA). It can spread from cell to cell to infect the whole tobacco plant. If TMV is genetically modified, the novel genes are expressed in infected tobacco plants.

Biofilm in sewage treatment

Trickle filter beds are used for sewage treatment. Biofilms form on the surface of the rock fragments. They contain decomposers which digest organic matter in the sewage into inorganic compounds such as ammonia, and also nitrifying bacteria that convert ammonia into nitrates.

Detecting tumours

Tumours can be located using tracking experiments with fluorescent (luminescent) probes.

Tracking experiments to diagnose cancer

Tumours cells have more receptors for the protein TRANSFERRIN in their plasma membranes than normal body cells. So fluorescent dyes are attached to transferrin and tumour cells in the sample are revealed by fluorescence of the cell surface. This method is especially helpful to diagnose hard to detect cancers.

What is gram staining?

Used to identify the 2 types of bacteria- those with and those without an outer layer of lipopolysaccharide and protein.

Production of Hepatitis B vaccine using TMV Pt. III: Benefits

Vaccination programs are difficult in remote areas because of problems with access and refrigeration of vaccines. Dried tobacco is also an easier way to administer the vaccine than a sterile injection of a liquid vaccine that needs to be refrigerated.

Continuous culture

When nutrient and waste levels can be adjusted to maintain the reaction indefinitely. The microbes are maintained at peak growth rate (exponential phase). The product is harvested continuously. All conditions are kept as constant as possible. It is more economical as smaller tanks are used. If the fermenter is contaminated, very large losses occur.

Batch culture

When the fermentation reaction is temporarily halted to replenish nutrients and remove waste products. The microbes go through all the stages of growth prior to collection of product. The products is separated from the mixture at the end. Conditions inside the fermenter change (temp. is monitored). Less cost-effective as larger tanks are used. If fermenter is contaminated, only one batch is lost i.e smaller losses.

Knowledge stored in a database increases ...

exponentially i.e. doubling every 18 months

3. Vector delivery genes

i. A. tumefaciens - a bacterium that inserts a plasmid into plant cells with the target gene. ii. TMV - a virus that inserts RNA into plant cells, with the RNA including the target gene.

1. Chemical methods

i. Calcium chloride - cells incubated in a cold calcium chloride solution take up DNA when given a heat shock. ii. Liposomes - artificial vesicles containing the DNA fuse with the cell membrane of protoplasts.

2. Physical methods

i. Electroporation - electric fields causes pores to open briefly in membranes so DNA can enter cells. ii. Microinjection - one micropipette holds the cell while another injects DNA through a tiny needle. iii. Biolistics - tiny metal balls with coated with DNA are fired at the plant to penetrate cells.

BLAST searches can ...

identify similar sequences in different organisms

PCR to detect different strains of influenza virus

it is a retrovirus, i.e. uses RNA as genetic material. RNA is extracted from the cells taken from an infected patient. This will include virus RNA and the patients own mRNA. Reverse transcriptase is used to produce cDNA copies of all the RNA in the sample. Primer samples specific to the strain of influenza being tested are added and PCR is carried out. If the strain was present, many copies of double-stranded DNA are produced, with viral base sequences on one of the strands. Fluorescent dyes that bind to the double-stranded DNA can be used to detect this DNA.

Genetic modification can help crops overcome...

unfavourable environmental conditions like: - providing resistance to herbicides - enabling growth in extreme conditions (saline/ dry) - resistance to pathogens/ viral infections It can also help increase the yield of crops.