Biotechnology Notes Finished PDF

lOMoARcPSD|8955794 BIO 154 Biotechnology Human life sciences (Universiteit Stellenbosch) Studocu is not sponsored or endorsed by any college or university Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|89557...

lOMoARcPSD|8955794 BIO 154 Biotechnology Human life sciences (Universiteit Stellenbosch) Studocu is not sponsored or endorsed by any college or university Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 BIOTECHNOLOGY Lecture One Vocabulary Hydroponics​: ​Process of growing plants in mediums with added nutrients but without soil. Biotechnology Examples of Biotechnology Introduction 1. Purple tomatoes - these contain high levels of anthocyanins which are healthy. These have been shown to reduce cancer levels in mice. 2. Sterile mosquitoes can help reduce populations and spread malaria and dengue less. 3. Salmon containing increased growth hormone levels allows for efficient farming. Biotechnology must remain ethical at all times. What is Biotechnology? This is the study of “life” and “tools”. Biotechnology​ is the characterization, use and change of orgaisms, using technical processes, to benefit mankind and the environment. History of Biotechnology This is no new concept, it has been used since the artificial selections of wolves began in 30,000 BCE. Why Biotechnology is Increased Populations Necessary When populations increase, more crop yields need to be produced for food and medicines need to be increased in availability and improved. It has been estimated that conventional plant farming will not yield enough food for the increasing population. Artificial Corn This is easy to peel. Steam cooks quicker. ~200 varieties. Can be grown in 69 countries opposed to natural corn grown only in central America. General Impacts of Biotech Reduction in the need for pesticides. Increase in crop yields. Increase in Farmer Income. People will live longer as medical technology improves and thus better medicines must be created. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Relevance of Amish Farmers Biotechnology: Examples Amish farmers grow nicotine-free tobacco and blight resistant potatoes. They earn twice as much with their biotech tobacco due to how unconventional they are. Fossil Fuels Biofuels are a newer development made from substances such as maize. However, this may lead to an increase in the food crisis as crops will take up space for fuel production and not food production. Stem Cells Biotechnology is responsible for stem cell development. DNA Technology This is used to identify both humans and plants. This is useful for legal purposes. Types of Biotechnology 1. Different Generations A. Conventional use Natural plant products, breeding/improvement, aspects of modern agriculture (hydroponics), fermentation. B. Use of ​in vitro ​technology Plant tissue culture, embryo rescue (plants) and ​in vitro fertilization (animals), bioreactors. C. Molecular plant biotechnology (DNA) Molecular markers, cloning and stem cell research, genetic engineering. 2. Different Colours Image: Lecture Notes Generations: First Breeding Generations Breeding and selection is an old practice used in which genetic Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 variation is selectively utilized. Biotechnology: Green Revolution Father is Norman Borlaug. Started in the 1950’s. Refers to the exponential increases in the yields of crops due to dedicated breeding and selection programs and improved agricultural practices. Natural Plant Products Farmers can sell the raw products (ginger roots/plants), the actual product (ginger) or extract the important substance from the product (ginger essence). As the previous list continues, more money is made. Fermentation Baker's yeast has been used since ~6000BC for beer brewing, wine making and bread baking. This yeast is very important for industries around the world. Generations: Second In Vitro​ Technology Generation In vitro​ means in glass. This includes all techniques in which plants/tissues go through a sterile culturing phase. Image: https://www.researchgate.net/figure/Main-technologies-used-in-assisted-reprodu ctive-technology-ICSI-intracytoplasmic-sperm_fig2_333366160 Generations: Third Molecular Markers Generation A molecular marker is a molecule which can be linked to a specific genetic trait. These are used to predict the phenotype of individuals by analyzing it on a DNA-level. Genetic Manipulation This is the (non-sexual) addition of genetic material to an Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 organism. These new genetic traits can be added to an organism or unwanted traits can be removed. GMO’s GMO = Genetically Modified Organisms This can occur naturally and done artificially. Natural Examples Sweet potatoes contain genes inserted using the same bacteria that humans use to make GMO’s. Some butterflies contain genes injected into them by parasitic wasps. There are very 0 deaths from GMO consumption by humans, it is very safe. Current Status of GMOs GMO’s are planted all over the world and in 2017, 17 million farmers in 28 countries planted 190 million hectares of GM crops. The first GM crops were released in 1996 and by 2005, there were already nearly 100 million hectares planted in more than 21 countries worldwide. Biotech Crops Four Main GM Crops 1. Soybean 2. Maize 3. Cotton 4. Canola Bt protein kills pests. This protein is placed into maize in South Africa and cotton, of these crops and therefore less pesticides are necessary. Are These Crops Beneficial to Developing Countries? If the quality of crops are low, the developing countries are burdened. South African “To embrace biotechnology is to further u=embrace our Biotechnology Strategy commitment to the realization of our national imperatives and specifically: 1. To improve access to and affordability of healthcare. 2. To provide sufficient nutrition at low cost. 3. To create jobs in manufacturing. 4. To protect and cherish our rich environment.” Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Lecture Two Vocabulary Codon​: Three bases (A,T, G or C) together. DNA Ligase​: ​A​n enzyme which can connect two strands of ​DNA together by forming a bond between the phosphate group of one strand and the deoxyribose group on another. Plasmid​: A structure containing genetics in a cell that can replicate without the use of the chromosomes, usually a small circular DNA strand in the cytoplasm of a bacterium. Taq DNA:​ This is the common enzyme used in the last step of the PCR. Taq DNA polymerase is a recombinant thermostable DNA polymerase and unlike normal polymerase enzymes, Taq DNA is active at high temperatures. Central Dogma The central dogma is a process in which the instructions given out by DNA are converted into a functional product (protein). DNA is a double stranded nucleic acid containing four bases, Adenosine, Guanine, Cytosine and Thymine. Three Processes of Central Dogma 1. Replication This is the process where identical DNA molecules are made. 2. Transcription This is the process where RNA is made using DNA as a template. 3. Translation This is the process where protein is made using mRNA. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: https://en.wikipedia.org/wiki/Central_dogma_of_molecular_biology Image: Source Unknown Codons Start Codon These codons present in mRNA stimulate the binding of a transfer RNA which ​starts​ protein synthesis. The start codons mark the site where translation begins. Example: AUG codon for methionine is a start codon Stop Codon These codons present in mRNA do not code for any amino acids and signal the end of the polypeptide chain in translation. The stop codons mark the site where translation ends. Stop Codons: TAA, TAG, TGA. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 DNA Manipulation DNA manipulation is done in via main methods such as: using restriction enzymes, Restriction Enzymes These enzymes cut a specific sequence of DNA. Example of cutting: Image: Source Unknown By doing this, separation happens on the strand where the cut occurs. The bacterial site where the DNA is specifically cut is called a “Restriction Site”. A restriction enzyme usually makes many cuts - making many restriction fragments. The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary sticky ends of other fragments. DNA Ligase​ is used to seal the bonds between restriction fragments. DNA Ligase Definition: A​n enzyme which can connect two strands of ​DNA​ together by forming a bond between the phosphate group of one strand and the deoxyribose group on another. Plasmid Plasmid: A small, circular piece of DNA that can replicate independently of the chromosome. The expression of the inserted gene will be regulated by the promoter. Image: https://en.wikipedia.org/wiki/Plasmid Plasmid Map A graphical display of plasmids and how the process occurs. The locations of major landmarks on DNA such as restriction enzyme sites, gene of interest, plasmid name, etc is shown on these. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: https://reflectionsipc.com/2017/04/24/promiscuous-plasmids-a-rapid-reflection-f rom-eccmid-2017/ Components of a Plasmid Image: Lecture Notes Obtaining Recombinant Recombinant DNA Molecule: Molecules of 2 different species DNA Molecule that are inserted into a host organism to produce new genetic combinations. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: Lecture Notes Bacterial Gene Cloning Plasmids are used to copy DNA. Process 1. Plasmid is isolated and cut with restriction enzymes (EcoR1). 2. Pieces of DNA are ligated from the chromosome into the restriction site of the cut plasmid using DNA ligase. 3. This plasmid with the new DNA is placed into a bacterium (e. coli). 4. This e.coli can be inoculated in liquid media and allowed to grow overnight. 5. The next day there will be a thick culture present. Each of these cells will contain a plasmid with the ‘cloned’ DNA. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: Lecture Notes DNA Determination There are multiple ways to determine DNA: 1. Polymerase Chain Reaction Only works if you know what the sequence of the DNA is. 2. Libraries These can be used to identify genes based on DNA similarity to other genes. Polymerase Chain Definition: ​A ​method used to rapidly make many copies of a Reaction specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. (Up to millions and billions in amount). This allows for rapid amplification of specific pieces of DNA but only if the DNA sequence is known via genome sequence or someone else identifying the gene. Note: PCR only works on one strand of DNA so DNA must be split in step 1. Components of PCR The technique relies on the lacZ gene, which encodes an enzyme called beta- galactosidase. This enzyme can be split into two parts: - The bacteria have one part of lacZ (called the omega fragment). - The plasmid has the other part (called the alpha fragment). When the bacteria take up a plasmid with an intact alpha fragment, the two parts work together to form a functional enzyme, which can break down a substance called X-gal, producing a blue color. 1. No Insert (Non-recombinant Plasmid): The bacteria produce a functional enzyme, turning colonies blue. 2. With Insert (Recombinant Plasmid): If DNA is inserted into the alpha fragment, it disrupts the enzyme, resulting in white colonies because X-gal isn’t broken down. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: Source Unknown PCR: Polymerase Chain Reaction There are 20-40 cycles of the same 3 steps. Each cycle results in doubled DNA strands and therefore many are produced. 1. Denaturation Double stranded DNA is split by heating it up to 94℃ for 1 minute. 2. Annealing The primers bind specifically to either side of the DNA being worked with. Usually between 50-60℃. 3. Extension Enzyme, Taq DNA polymerase, elongates the DNA from where the primers are present which continues at 72℃ for 2 minutes. Image: https://en.wikipedia.org/wiki/Polymerase_chain_reaction Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: https://www.khanacademy.org/science/ap-biology/gene-expression-and-regula tion/biotechnology/a/polymerase-chain-reaction-pcr?modal=1 Image: Lecture Notes Genome Libraries Libraries are used to identify genes based on DNA similarity to other genes. Types of Libraries 1. Genomic Contains randomly cut out pieces of DNA which are isolated from the nucleus. If these DNA pieces are from a eukaryote, then both exons and introns are present. 2. Metagenomic Contains randomly cut pieces of DNA isolated from an environmental sample. 3. cDNA Contains DNA synthesized from RNA using the enzyme reverse transcriptase. No introns are present. Production of a Genomic DNA Library 1. DNA is cut by restriction enzyme digestion (eg: EcoR1). Many fragments of different DNA pieces are then available. 2. Vectors (plasmids) are cut with the same enzyme. 3. Ligate the pieces of DNA into the plasmids using DNA Ligase. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 4. This results in many plasmids with different DNA. 5. These plasmids (millions) are placed into tubes. Image: https://www.britannica.com/science/genomic-library Genomic DNA Gene Structure: A gene consists of ​regulatory domains (promoter, activator, silencer and terminator sequences) and an open reading frame​ (ORF) that contain exons and introns. Image: Lecture Notes Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Regulatory Domains Activator: P​ rotein (transcription factor) that increases gene transcription of a gene or set of genes. Silencer: D​ NA sequence capable of binding transcription regulation factors. Promotor: D ​ NA​ sequence that defines where the transcription of a gene by RNA polymerase begins. Terminator: ​DNA​ sequence that defines where the transcription of a gene by RNA polymerase ends These are used by transcriptional machines to determine where these genes will be transferred. Eg: only transcribed in the liver. Constitutive Gene: Gene transcribed everywhere in the body at all times. Problem with Cloning The identification of bacteria containing plasmids with an insert poses a problem as it is difficult to determine if a bacterium contains a plasmid with an insert or not. Alpha - Complementation Special cloning vectors (plasmids) containing bits of the lac ​operon contain part of the gene for beta - galactosidase (also known as lacZ). This beta - gal (lacZ) catalyses the degradation of the sugar lactose. It can also use an artificial substrate (eg: X-gal) and when X-gal is degraded it produces a blue dye. This helps scientists as the colonies which stain blue do NOT contain an insert whereas the colonies which stain white do contain an insert as it will be unable to produce an active lacZ protein. Note: The other half of lacZ needed is present in the organisms’ chromosome. Image: Source Unknown Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 What This Looks Like on A Petri Dish Image: http://bio3400.nicerweb.net/Locked/media/ch19/Xgal.html DNA Library Uses Genomic DNA libraries can be used for recombinant protein production of: Protein hormones (EG: insulin) Enzymes for washing powders Vaccine epitopes Molecular biological reagents Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Lecture Three DNA Synthesis DNA is synthesised naturally in a cell, but biotechnology has allowed for DNA to be replicated on command. Note: Primers are usually 50bp long with a 20bp overlap. Making DNA 1. Oligo Synthesis. 2. DNA synthesis. Extension PCR fills in the gaps between the primers. Larger DNA molecules can then be assembled together using a similar extension PCR process. Eventually, the entire gene can be amplified by the PCR using primers specific to each end. Advantages of Creating DNA With This Method 1. Does not need template DNA (advantage over with PCR). 2. Can optimise DNA sequence for a particular organism to help enhance expression. How This Method Is Used Proteins have been stained with dichromatic blue. No Induction:​ A promoter is induced by adding a chemical. No protein beta is made. Native Construct:​ Gene has been amplified with PCR. Codon Optimised:​ E. coli can make more RNA when the codon is optimised. More protein is therefore produced. Image: Lecture Notes Artificial Life Artificial life is being created using this technology. Mycoplasma mycoides​ was synthesised (1000 0000 bp genome) and transplanted into ​Mycoplasma capricolum​. Used the designed nucleotides to make up large pieces of DNA that make up different sections of the genome. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 E.coli ​and ​S. cerevisiae​ was used to make these large bits of DNA into full, complete genome. ​Mycoplasma capricolum​ was removed of its DNA and replaced with the synthesized DNA. It was shown that the ​Mycoplasma mycoides​ was expressed. Image: Source Unknown Minimal Genome Project This is a project in which people are trying to identify the smallest amount of DNA necessary for bacteria to survive. A group examined ​Mycoplasma genitalium​, which (when they started the project) had the lowest number of genes, 475, known in a microbe. Since then, however, some ​Candidatus​ species have been identified with fewer than 200 genes. The group found that 372 genes are necessary for a bacterium to survive. They did this by removing one gene at a time and analysing if Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 the change was lethal. The minimal genome has been synthesised and transplanted into cells containing no DNA leading to the first artificial organism ​Mycoplasma laboratorium​. How Genomes That 1. Transgenesis​. Already Exists Are Altered This is when a new piece of DNA (artificial) is added to the genome at a random place. 2. CRISPR/CAS9 Technology that alters the genome at a specific defined place. Transgenesis: Transgenic Organism: ​This is an organism that contains a set Overexpression of genes from another organism which has been artificially placed into its genome. Overexpression: ​This adds a new activity or increases the amount of one already (enzyme activity) present. Introducing a new activity: want something novel/new. Example: wanting the organism to make a new chemical which can be used as a drug. These constructs are made in the plasmids (circular bits of DNA). Ligase and restriction enzymes are used to put them in the order of the below image. Note: ​This is ​sense constructed​ as the gene is in a sense orientation. The start codon is just after the promoter and the stop codon is just after the terminator therefore, this can be read from left to right. Image: Lecture Notes Examples of Overexpression 1. Mice grow obese if they are overexpressing PEP carboxylase. This tells us this gene is important in the process of fat distribution. 2. Spider silk can be expressed in animals. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Transgenesis: Antisense Antisense and RNAi:​ These ​repress​ the expression of a gene and RNAi present in an organism. Antisense In this case, the gene is in an antisense orientation. The gene has a start codon after the promoter and a stop codon before the terminator = antisense orientation. If you place this in an organism, it will create antisense RNA. Double stranded RNA will form and the organism will naturally start degrade the RNA strand (RNA viruses are often double stranded). The antisense and sense strand will be removed. The sense RNA is therefore removed and will not be able to make protein. RNAi Two bits of the gene are identical but opposite directions: one is sense orientation and the other is antisense orientation. They are separated by spacer DNA. When the organism begins transcribing, the sense RNA will be transcribed from the ATG after the promoter, through to the ATG before the terminator. The RNA will go into the cytosol, and the two bits of gene are identical but mirror images and therefore they will naturally align and form double stranded RNA - therefore it will be degraded. RNA is cut into 23 nucleotides which will bind to the native sense RNA (that you want to remove). This will then be degraded (the sense RNA). Image: Lecture Notes Antisense and RNAi Example Polyphenol oxidase (PPO) causes apples to go brown. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 This PPO is removed and therefore stops apples browning. Cisgenesis Cisgenic Organisms:​ Organisms that have been engineered using a process in which genes are artificially transferred between organisms that could breed (closely related organisms). Cisgenic plants only use DNA from that same plant. Same Apple Example: Promoters, genes and terminator sequences from the apple to make an antisense construct is transformed back into the apple. Image: https://www.wikiwand.com/en/Cisgenesis CRISPR/Cas9 This is a new way to manipulate genomes. CRISPY and Cas9 are bacterial genes that help protect the bacteria against infection by viruses by recognizing and cleaving viral DNA. Scientists found little areas which contained viral genes in bacterial DNA. They worked out that these are defense mechanisms. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 If the bacteria is invaded by a virus, it transcribes these CRISPR loci which contain these viral sequences. These will identify and bind to the viral DNA. They will guide enzymes (cas9) to cut up the DNA at those specific points. This stops the virus from attacking the bacteria. Utilizing CRISPR RNA is placed into the organism along with Cas9. This RNA will bind to a specific site and the Cas9 will bind to the RNA and cut the DNA (chromosome) at the specific site. The repair mechanisms will attempt to ‘glue’ the chromosome back together. Base pairs are added/removed when this occurs, resulting in a mutation​. It will therefore be ​unable to produce a protein​, this protein is therefore removed from the organism. Image: http://sitn.hms.harvard.edu/flash/2014/crispr-a-game-changing-genetic-engine ering-technique/ Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: https://www.cambridge.org/core/journals/mrs-bulletin/news/crispr-implications-f or-materials-science Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Lecture Four Microbes The total biomass of life on earth is estimated to be 450 billion tons. Of this, 280 billion is microbes (about 60%). Microorganism Characteristics Microorganisms grow exponentially. They have very short generation times. Example: A single cell of E. coli can generate 500cells within 3 hours and 20 mins. Limitations to Growth Availability of nutrients may be depleted. As they grow, they produce toxic compounds which build up to remove competition in their environment. Image: https://www.thoughtco.com/bacterial-growth-curve-phases-4172692 Diseases Microbes can cause diseases that may outbreak into pandemics. Epidemic:​ Localized area. Pandemics​: Epidemics that spread over a wide geographical area (globally). Examples of Pandemics Spanish Flu Smallpox COVID-19 Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Biological Warfare What is biological warfare? Warfare that uses bacteria, viruses, etc, to kill people, animals, and food crops. First Case The first case of biological warfare was in 1348. A Mongol army invaded Europe and catapulted bodies of soldiers who died of plague into the city of Crimea. It is thought that this led to the black death pandemic which killed 75 million people worldwide and ⅓ of the European population. Smallpox Smallpox killed 300-500 000 000 people in the 20th century alone. 30% of infected individuals died. This virus was overcome by mass vaccination. The smallpox eradication programme was run by the WHO between 1966 - 1980. Vaccination was a major weapon to remove this virus from the human population. Smallpox Vaccination Closely related to smallpox and cowpox. Makes one feel slightly ill but become completely immune. Virus stocks are kept in two places in the world: the US and Russia. This is kept in case of a re-emergence from skeletons. Vaccines These are made using ​genes from the disease​ ​causing agent.​ These are expressed in a non-pathogenic organism. This means that there is no possibility of infection when the vaccine is used (antibodies are produced). Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: Source Unknown Herd Immunity Herd Immunity When enough people are vaccinated in a community, the rest of the population has a lower chance of becoming infected. Benefits Stops the spread of the disease which helps people who cannot be vaccinated (immunocompromised people). Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: https://www.wildcat.arizona.edu/article/2020/03/n-herd-immunity Biological Warfare Cont. Methods to Make Disease More Dangerous 1. More infectious 2. Resistance to antibiotics 3. More deadly 4. Overcome immunity from current vaccines Gene Shuffling Method for changing the genes of organisms via single gene shuffling or multi gene shuffling. Two Types 1. Single Gene Shuffling Individual genes (nucleotides) are altered using chemicals which change amino acids therefore changing proteins. This may result in them becoming more resistant to antibiotics. 2. Multigene Shuffling This is more powerful. Scientists cut up the genes and ligate them together again randomly. This results in resistant genes in each separate DNA. This multigene shuffling results in a much larger resistance to antibiotics. Image: https://bitesizebio.com/29721/dna-shuffling-like-pro/ Virus Recreation The smallpox virus genome has been sequenced and is available in sequence libraries. It can therefore be recreated. However, important bits of the genome sequence are likely to be left out. In 2002, the Polio virus was created in a lab by synthesising its genome and allowing it to assemble. Insulin The first protein produced by the pharmaceutical industry using recombinant DNA technology was insulin. Before synthetically produced insulin, insulin was produced from pig pancreas. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 2000kg of pig pancreases produced roughly 250g of insulin. The process of producing this insulin includes a large scale industrial plant involving fermenters, centrifuges, purification columns, etc. Disadvantages This was expensive and not easily available. Some people also developed a resistance to non-human insulin. Malaria Malaria Statistics in 2010 200 000 000 cases. 650 000 deaths. Malaria is ​treatable​, but there is no vaccine available. However, Artemisinin is antimalarial. Artemisinin Artemisinin can be found in plants, but the cultivation and extraction process is very long. It can be up to 14 to 18 months. A company called Amyris developed a new production method using synthetic biology to produce Artemisinin. Two Stage Processes Stage 1:​ Production of artemisinic acid in ​Saccharomyces cerevisiae​. Stage 2: ​Chemical conversion of artemisinic acid to artemisinin. Advantages of Stage 1 This is faster (months) whereas stage 2 is slower (years). Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Lecture Five Plant Products Uses of Starch Thickener in the food industry. Makes paper feel smooth. Lubricate tight fitting rubber clothing. One of the main ingredients used to produce bioethanol. Can also be used to make biodegradable plastic plastic. Uses of Oil Food and cooking. Recreational purposes (essential oils for relaxation). Convert into fuel (biodiesel). Biodiesel Plants Used for Biodiesel Plants which don’t produce oil as a major compound (oil = byproduct). Soybean and corn. Canola is also used too. Image: https://pubs.rsc.org/en/content/articlelanding/2019/gc/c8gc02698j#!divAbstrac t Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: Lecture Notes Algal Use for Biodiesel Advantages: This produces relatively high amounts of oil; can grow in saltwater; doesn’t need soil. This, however, is still too expensive therefore in the future yields will need to be improved. Protein Protein is another plant commodity. Having the correct balance of amino acids is important for good health. Some staple foods (maize) lack specific amino acids. Issues When Relying on Plants for Protein Legumes lack methionine. Maize lacks lysine and tryptophan. International Maize and Wheat Investment Centre This company focuses on increasing the amounts of amino acids, lysine and tryptophan, into maize. Children who consume QPM (Quality Protein Maize) benefit from 12% increased weight and 9% increased height. Plants as Bioreactors (F Plantibodies These are antibodies made in plants by transforming the animal gene for the antibody, into plants. Advantages Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 The protein folds correctly, which does not happen if the same gene is transformed into microbes. Plant Vaccines Vaccines can be made in plants by expressing an epitope from a disease causing organisms. Advantage over doing this in microorganisms is higher yield. Plant Cell Walls Plant Cell Wall Uses Paper Clothing Bioethanol & Plant Cells Plant cell walls cannot be used to produce bioethanol as it contains lignin, which makes it difficult for the substance to undergo fermentation. This can be corrected by genetically engineering plants to produce less lignin. Image: Source Unknown Starch and Oil These are derived from the bits of the plants that are edible. These are able to be used for bioethanol. Starch, oil, protein and cell walls are all examples of​ high volume, low price commodities. But plants can also produce​ low volume, high price products​. However, some of these can be synthesized ​artificially​ which lowers the cost but others are too complex to synthesize. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Example: Taxol Taxol is a product of secondary metabolism. Taxol​ ​inhibits mitosis. It is used in breast cancer treatment. It is found in the bark of the Pacific yew, one of the slowest growing plants in the world. It would take eight 60 year old trees to extract enough taxol for ​just one patient​. This is therefore not commercially viable. The market for taxol is more than 1 billion US dollars per annum. Plant Cell Bioreactor These are used to grow the plant cell cultures quickly compared to how slowly the plant cells would grow in the Pacific yew. Process 1. Cell cultures are isolated from Pacific yew. 2. These are placed into bioreactors. 3. It is fed with nutrients and matures. Image: https://link.springer.com/referenceworkentry/10.1007%2F978-3-642-22144-6 _123 Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Lecture Six Vocabulary GMO:​ Genetically Modified Organisms Callus: ​Soft tissue that forms over a wounded/cut plant surface, for healing purposes. Totipotent:​ Plant cells that can regenerate into any plant cell. Genetically Modified Plants Are GM Plants Dangerous? It is estimated over 100 billion animals have eaten GM plants with zero reports of adverse effects over 18 years. Example: Watermelon Traditional Breeding: Planting and growing seed. Beneficial mutation may occur and that plant will be favoured and those seeds from the mutated plant will be used. GM: New DNA is introduced into the genome of the watermelon. Image: https://jameskennedymonash.wordpress.com/2014/07/14/artificial-vs-natural- watermelon-sweetcorn/ Benefits of GM Plants These provide bigger yields of plant products. This is necessary as the word population is increasing exponentially, resulting in food security being threatened. The larger yields of crops ensure that the population has enough food. It has been analyzed by scientists that GM crops would increase yield by 5-24%. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 They also found that GM plants contained fewer toxins caused by fungal infections. Image: https://www.quora.com/What-is-the-Malthusian-theory-of-population-growth Types of GM Types of GMOs Herbicide resistant​ - makes farming more efficient. Insect resistant​ - reduces use of toxic insecticides. Drought resistant​ - helps overcome impact of climate change. Improved bioethanol production​ - increases efficiency. Non browning apples​ - decreases food wastage. Plantibody production How Plants Genetically Step One: Make Callus Modified A callus is an unspecialized cell. They are totipotent (can regenerate into any plant cell) and can regenerate an entire plant from one callus cell. 1. Sterilised plant material is placed onto media containing hormones. The rate of cytokinin to auxin can alter how the plant reacts. Step Two: Trans DNA Preparation 2. Bacterial selection is used while making the construct. A selectable marker helps identify transformed cells. The promoter expresses the trans genes. The trans gene is then made into RNA, and will introduce a new activity/repress gene activity. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: Source Unknown Step Three: Plant Transformation This is the direct transfer of genetic material into a living cell. 3. Two Transformation Techniques: Agrobacterium ​mediated transformation:​ Ti plasmid is sequences, restriction enzymes cut out genes that cause gall disease, introduce multiple cloning sites, bits of DNA are then placed inside plasmid using DNA ligase. This plasmid is placed back into ​agrobacterium which is then used to transform callus. From callus cells, new plants are grown. Direct Transformation Techniques:​ For intact plant cells, biolistics is used to shoot the trans-DNA into the cell. Use tungsten or gold bullets as carriers of the DNA. For intact plant cells: Biolistics shoot the trans-DNA into the cell. Use tungsten or gold bullets as carriers of the DNA. Step Four: Selection Not all cells will become transformed (Less than 1%). 4. The selectable marker gene helps identify the transformed cells. It can lead to resistance to antibiotics, herbicides, high salinity and toxic sugars. * Take note of the Selection Genes section below. Step Five: Plant Regeneration This normally runs concurrently with selection. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 5. The transformed cells are placed onto media and regeneration is allowed to occur. Hardening off​ occurs - allows plants to start controlling their water relations after tissue culture. Step Six: Identifying Individual Plants where The Transgene is Effective 6. The final step is selecting for the plants where the transgene is having the effect that you are looking for. From a dozen plants, search for transgene expression or reductions in RNA. Selection Genes Without Selection The untransformed cells will grow at the same time. Vast majority of plants will be untransformed. Very few will be transformed. Very labour intensive - to identify transformed plants. Chimeras will be present - these are when cells with different genetic makeups are present in the same organism. With Selection Only the transgenic bits grow therefore all plants will be transformed. However, due to many plants being killed off, there will be few plants. Image: Source Unknown GM Plant Regulations: RSA Permits issued in SA between 1999 and 2014 Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: Source Unknown GMO Permit Application Process 1. Application including field trials in other countries, in SA, toxicity compounds produced by plants, etc is presented to the department of agriculture, forestry and fisheries where a report will be written. 2. The report will then be handed to the advisory committee where they will scientifically evaluate it. 3. The executive council then obtains this report and they make the final decision on whether to grant the permit or not. Regulation of GM Crops in SA These aspects will be tested. Allergenicity Toxicity Changes in composition Yield Note: GMO crops must be shown to be functionally equal to non GMO crops. Conventional Plants Normal plants undergo ​no​ testing. This means there are no regulations and there is an assumption that these plants are safe - which isn't entirely true. The Lenape Potato This was a potato developed in the 1960’s and almost reached the market before it was found that the tuber contained TOXIC levels of solanine. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Lecture Seven Four Stages of Drug 1. Discovering drugs. Development 2. Pre-clinical trials: tested on animal models. 3. Clinical trials: tested on groups of humans. 4. Drugs are monitored when released to the public. Ethicality of Trials At Stellenbosch, there are 5 separate ethics committees. Any experimentation on vertebrates needs to be ​approved​ before the experiment can begin. The Declaration of Helsinki These are the ethical principles followed by medical personal revolving around medical research on humans. The human partaking in the clinical trial must be fully aware of the possible effects of the drug trial. Informed consent from humans ​must​ be acquired in a written form. Examples of Unethical Experiments Elephants were given large doses of LSD (acid). The elephants died from this. 57 children were fed oats laced with plutonium and they obliged as they were told they were taking part in a ‘science club’. AZT testing. This was the testing for a drug to alleviate HIV symptoms. Children (in foster homes) in New York were tested on and ingested AZT. Patients in Zimbabwe were also tested on with AZT. Discovery of Drugs Note: Often they are discovered accidentally. 1. Target Identification Drugs often react with cellular or genetic chemical molecules, known as ​targets​. These can be associated with a specific illness. Targets are therefore identified and its association with the illness is determined. 2. Target Validation Targets are compared to other possible candidates in terms of its association with a specific illness. Tests are done to ensure that the interaction between the target and drug leads to the appropriate chain in “sick cells”. 3. Lead Generation A lead molecule has the potential to treat the illness with. This includes comparisons with other known molecules to determine its potential. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 4. Lead Optimisation Compare the different lead molecules and choose the one with the greatest potential to be developed as an effective drug. This included ​in vivo​ (in live organisms) and​ in vitro​ (in cells) studies. Safety tests are carried out regarding promising molecules: ADME: Absorption, Distribution, Metabolism, Excretion, Toxicity. Process of Discovering and Releasing a Drug. Image: Source Unknown Pre-Clinical Trials Pre-Clinical Trials Potential drugs with the necessary modifications are tested on non-human organisms to see how the drugs affect the organism. These are generally animals, however, certain animals are not closely related to humans therefore testing on them would not provide an accurate representation of the effects the drug would have in humans. Example: Chocolate is fine for humans but toxic to dogs. The Alternative to Using Animals Human Organs-on-Chips: see chip below. Cell cultures (animal cells grown in liquid culture) which are exposed to the drug. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: https://www.theguardian.com/artanddesign/2015/jun/22/the-end-of-animal-te sting-human-organs-on-chips-win-design-of-the-year Image: https://www.hepb.org/blog/phase-3-clinical-trials-opening-hepatitis-delta-pati ents/ Clinical Trials Clinical trials This is the study of drug development and ensuring its safety in humans. Drugs are tested on human control groups. Organizations such as ethics committees must give clearance before clinical trials can commence. Clinical trials can only commence if no adverse or damaging effects were seen in the pre-clinical trials. Phases of Clinical Trials Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: Lecture Notes Placebo Effect: ​Occurance in which some people experience a benefit after the administration of an inactive "look-alike" substance or treatment. Pharmaceutical Pipeline of This process takes from 10-15 years in total. It is a long and Drug Development thorough process. This explains why it takes so long and is so expensive to create/discover a new drug. Drug companies must therefore make their money back and this is why new drugs cost so much. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: Lecture Notes Difference Between a Treatment Treatment and Vaccine This can help cure a disease once infected. Vaccine This prevents infection. Example: Smallpox vaccine has led to the elimination of smallpox in the wild. Example of a Treatment vs A Vaccine Image: https://www.vumc.org/viiii/spotlight/antibody-therapy-vs-vaccine Coronavirus Treatments Dexamethasone​: Anti-inflammatory used to treat patients on ventilators. Blood plasma​: Taken from people who have recovered and should contain antibodies that bind the coronavirus. Hydroxychloroquinine: ​Evidence is that it is ineffective. Vaccines Current record for fastest found vaccine: 4 years. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 There are currently 24 vaccines undergoing human trials: 5 are in phase 3, 6 in phase 2, the rest are in phase 1. Russian Sputnik 5 Vaccine: 2 coronavirus genes have been transferred to an adenovirus. Coronavirus proteins are produced by the adenovirus. This has been expedited to phase 3 trials. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Lecture Eight Vocabulary Stem Cells:​ A cell with the unique ability to develop into specialised cell types in the body. Truncated​: Shortened. Organ Rejection:​ When the immune system of the patient recognizes the donor organ as foreign and attempts to eliminate it. Plasticity​: The adaptability of an organism to changes in its environment between its various habitats. Bioreactors​: They are systems or devices that support a biologically active environment. Gene Therapy Gene therapy is a method used to cure genetic diseases. This is done by ​modifying​ the ​genome​ of an organism to treat or prevent genetic diseases. Two Gene Therapies Currently Available in US 1. Treats a form of leukemia 2. Curse a rare form of blindness: replaces the gene that causes blindness. This treatment is very expensive. Duchenne Muscular This disease affects 1 in 5000 boys and it is generally fatal by Dystrophy (DMD) the age of 30. Truncated Gene: The people with this gene are still unaffected. Gene Deletions: This leads to the disease becoming present. Note: Golden retrievers can also suffer from DMD. They were therefore used to test drugs for this disease. These dogs can be cured using the mini-dystrophin gene (a bit of gene that is still active) which was placed into the genome of a virus. This was then injected into the dog which led to the protein being made. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: Lecture Notes Stem Cells Stem Cells: These cells are able to develop into specialized cells. Image: https://www.yourgenome.org/facts/what-is-a-stem-cell General Notes on Stem Cells Most body cells are specialised to perform particular functions, such as red blood cells which carry oxygen around our bodies in the blood. Red blood cells are unable to divide. Stem cells provide new cells for the body as it grows, and replace specialised cells that are damaged or lost. Three types of stem cells: embryonic stem cells, adult stem cells, induced pluripotent stem cells. Advantages of Stem Cells No issue of organ rejection (see vocabulary) if grown from your own stem cells. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: https://www.thoughtco.com/pros-and-cons-of-stem-cell-research-375483 Classification of Stem Cells Stem cells can be classified according to their plasticity. Totipotent​ cells give rise to all embryonic and extraembryonic cell lines. Pluripotent​ cells can produce all embryonic cell types. Multipotent​ cells differentiate to a great number of cell types depending on the liquid media they are submerged in/nutrients they are exposed to. Totipotent Cell This cell has the ability to differentiate into different tissues, organs and even organisms. Can regrow an organism from these cells. Pluripotent Cell This cell has the ability to differentiate into a specific organ or tissue. Example: A new tail grows on a lizard. Multipotent Cells This cell has the ability such that of a pluripotent cell to develop into a specific organ (in the correct nutrient media). Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: https://www.researchgate.net/figure/Traditional-view-of-stem-cell-hierarchy- The-totipotent-stem-cell-can-form-all_fig4_3245838 Image: https://www.mdpi.com/2077-0383/8/5/627/htm Ethical Issues Issue​: Utilizing cells from human embryos (ultimately killing them) which have the ability to become human life posed an ethical conundrum. Solution:​ Scientists have developed methods to convert somatic stem cells into pluripotent ones. three-dimensional structures that support cell growth and tissue formation in biology Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: https://sites.google.com/a/nv.ccsd.net/the-international-society-for-stem-cell -research/background Artificial Meat Artificial Meat: Meat is made efficiently in the laboratory. Several start-up companies have been focussing on working out how to do this commercially. Advantages Less effort. No livestock needed. Cheaper. Less greenhouse gas production. DIsadvantages May not be as ‘tasty’. Image: Lecture Notes Cloning Animals Cloning​ is a process that lets one exactly copy the genetic, or inherited, traits of an animal. Livestock species that have been successfully cloned include: cattle, swine, sheep, and goats. Note: Mortality rates of young cloned animals are extremely high, during both pregnancy and infancy. Advantages of Cloning Animals Improving animal herds through increasing numbers of animals with advantageous traits. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Conservation purposes: increase animal population size. May bring back extinct species e.g. woolly mammoth. (This may be very difficult due to the degraded DNA). Disadvantages of Cloning Current success rate is quite low. Nature of twins may be different - not identical. The donor cell needs to come from a living organism, therefore it will be difficult to clone extinct organisms. Note: First successfully cloned animal is Dolly, a sheep. This was a result of 277 attempts. Somatic Nuclear Cell Somatic cell nuclear transfer ​(SCNT) is a laboratory Transfer strategy for creating an embryo from a body cell and an egg cell. Technique The technique consists of taking an enucleated oocyte (egg cell) and implanting a donor nucleus from a somatic (body) cell. Process in Detail 1. DNA from the donor cell must be inserted into an egg. Egg is prepared by enucleation. 2. Pipette sucks out the nucleus. 3. DNA from the donor cell is inserted into the egg cell. 4. Embryo is transferred to a surrogate mother for gestation. Animals Which Have Been Cloned By SCNT: Sheep, deer, mice, rabbits, cattle, pigs, horses, dogs and cats. Image: https://en.wikipedia.org/wiki/Somatic_cell_nuclear_transfer Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: Source Unknown Embryo Twinning Embryo twinning​ is the formation of twins via the artificial splitting of an embryo at the cleavage or blastocyst stage. Technique The splitting embryos in half. This results in identical twins but the exact nature of those twins is the result of a mix of genetic material from two parents. Animals Which Have Been Cloned Via Embryo Twinning: Sheep, cattle, ape. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: https://www.timetoast.com/timelines/the-history-of-cloning-c3fff8ba-a533-4f 97-9521-facf083db749 Animal Transgenics Note: Cloned animals and transgenic animals are DIFFERENT. Transgenics​ are genetically modified organisms with DNA from another source inserted into their genome. These are generally laboratory animals which are used for research. A large amount of transgenic animals have been created such as mice, cows, pigs, sheep, goats, fish, frogs and insects. Goals of Transgenic Animal Creation Research into animal and human disease. Improve livestock animals. Use of animals as bioreactors. Animal Transgenesis Transformation Methods Retrovirus-mediated transgenesis. Pronuclear microinjection Introduces the transgene DNA at the earliest possible stage of development of the zygote/ DNA is injected directly into the nucleus of egg or sperm. Sperm-mediated transfer. Gene guns. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: https://link.springer.com/chapter/10.1007/978-3-319-92348-2_6 Transgenic Animal Examples Glofish These are fish that glow in the dark. The gene that causes fluorescence (GFP gene) in jellyfish was inserted into zebrafish (a fish that is normally just black and white). The glofish are on sale throughout America (except California). They retail for 5 dollars per fish which is ten times the price of zebrafish. Image: http://glofishs.blogspot.com/2013/03/gmo-glofish.html Enviropig Express phytase in their salivary glands. The phytic acid in the pig meal is degraded releasing phosphorus. This phosphorus is absorbed by the pig. Normally the phytic acid passes through the pig and is excreted as waste (leading to eutrophication). Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: Sources Unknown Image: https://www.discovermagazine.com/technology/meet-the-genetically-engine ered-pig-with-earth-friendly-poop Gene Targeting Gene targeting​ is a genetic technique that uses homologous recombination to modify an endogenous gene. Scientists inactivate a particular gene and observe the effect it has. This provides information as to what that gene actually does. Example: Knock-out Mice “Knock-out” technology allows for the specific loss of a gene in mice. This allows for the function of the KO’d gene to be deducted Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 from the defects seen in mice. This can be used to mimic some disease. These mice require embryonic cells. Image: https://en.wikipedia.org/wiki/Knockout_mouse Animal Bioreactors Bioreactors: ​A vessel in which a biological reaction or (Pharming) change takes place. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 "Pharm-goat" Produces How Transgenic Animals Are Used as Bioreactors antithrombin 1. Gene for a desired protein is introduced via protein (mutations in transgenics to the target cell. gene cause 2. By using cloning techniques, the cell is raised to blood clots) become an adult animal. Antithrombin 3. Produce milk or eggs that are rich in the desired was 1st agent from milk of protein. transgenic farm animals to be approved for Spider Goat human Nexia Biotechnologies transferred the silk gene from consumption) Orb spiders into goats. The resulting male goats were used to sire (cause of birth) silk-producing female goats. Each goat produces several grams of silk protein in her milk. The silk is extracted, dried to a white powder, and spun into fibers. The fibers are stronger and more flexible than steel. Negative Transformation The inserted DNA randomly integrates into the genome. The eggs must be harvested & fertilized ​in vitro​. More than one copy of the gene may get into the genome. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Lecture Nine Genome-Wide Association Genome-Wide Association Study (GWAS) Sequencing the genome of genetically deviant people and genetically normal people and comparing the results. This can be used to identify genes involved in.. 1. Genetic diseases, eg. some cancers. 2. Plant yield. 3. Animal meat quality. 4. and many more things. What is a genome-wide association study? Information: https://www.genome.gov/about-genomics/fact-sheets/Genome-Wide-Associa tion-Studies-Fact-Sheet Image: https://www.researchgate.net/figure/Genome-wide-association-studies-GWA S_fig3_328344689 Sequencing a Genome First human genome took 15 years and $40 000 000 000 to complete. Used a process called Sanger sequencing that sequences 1000 bp at a time. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 ‘Next generation sequencing’ NGS machines can sequence 13 000 000 000 base pairs overnight and cost less than $1000. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: Source Unknown Issue with GWAS Issues have risen with the information that is obtained from DNA sequencing with regards to GWAS. Issue A genome sequence can tell you what genes are altered, but not​ where they are expressed. Solution Using ​transcriptomics,​ which is the study of how proteins are expressed in relation to sequences that code for them. Transcriptomics Transcriptomics​: The study of transcriptomes and their functions. Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Transcriptomes:​ The set of all RNA transcripts, including coding and non-coding, in an individual or a population of cells. Transcriptomics Process The process of transcriptomics starts with the extraction of RNA. 1. Depending upon the experimental requirement either total RNA or mRNA is extracted from the tissues. 2. Using the reverse transcription PCR method, cDNA is constructed from the mRNA. 3. A special type of polymerase called reverse transcriptase-polymerase, constructs DNA from the mRNA. 4. Finally, the cDNA or complementary DNA is ready to be analyzed. Image: https://www.macrogen.com.au/ngs/ts/ Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: Source Unknown Mass Spectrometer Metabolites:​ These are the intermediate products of metabolic reactions catalyzed by different enzymes that naturally occur within cells. The metabolites extracted from muscles during biopsies are analyzed with mass spectrometers. Two Types 1. Gas Chromatography - Mass Spectrometer 2. Liquid Chromatography -Mass Spectrometer Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: https://www.chemguide.co.uk/analysis/masspec/howitworks.html Pharmacogenomics This is the study of identifying how different people respond to drugs based on their genetics. Image: https://link.springer.com/chapter/10.1007/10_2019_110 Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Image: Source Unknown Biological Processes GWAS​ is also used to study biological processes such as cancer infecting the body. Different types of cancers can be studied too, not just their development but their differences. This may help identify cures. “Omics” Technologies Examples Proteomics Genomics Metabolomics Sociomics Innomics These provide us with the necessary platform for functional analyses of genes and their effects. Most Important Ones (NB) Image: Source Unknown Downloaded by Danielle Luyt ([email protected])  lOMoARcPSD|8955794 Downloaded by Danielle Luyt ([email protected])