Biotechnology Applications

• Genetic Engineering

  Humans have been selectively breeding plants and animals for years – to produce the desired traits in these species that we want.

  • This includes animals and plants.
  • Allows great control than selective breeding.

  Genetic engineering is the process of manipulating/changing the genetic material/genome of an organism.
• Genetically Modified Organisms (GMO)

  • Recent genetic engineering techniques allows for transfer of DNA from one organism to another.
  • How are they able to achieve this?
  • Scientists can remove undesired genes, or add in desired ones.
  • Bacteria and yeast is used to manufacture bulk proteins (protein engineering).
• DNA Cloning

  • Requires DNA to be extracted from target DNA.
  • Uses DNA ligase and restriction enzymes, & fluorescent label probes.
  • Target DNA is put into a bacteria and then cultured -this clones the  bacteria/target DNA.
  • Bacteria undergo binary fission.
  • This is quicker and cheaper than artificially synthesising DNA.
  • Uses bacteria plasmids.
• DNA Cloning

  • Gene of interest is placed in plasmid.
  • Forms recombinant plasmid.
  • This is placed back in the bacteria.
  • Bacteria replicates, making more copies of the gene of interest.
  • Bacteria’s ribosomes can also synthesise the plasmid, making proteins which is then collected for human use.
  • A form of protein engineering.
• Protein Engineering - Yeast

  • Yeast has been used for processes such as baking, brewing and winemaking for centuries.
  • Have been used to manufacture specific proteins (as protein factories).
  • Often used to produce some antibiotics and antigen proteins for vaccines.
  • Some vaccines made using this method are to prevent:
  • Hepatitis B, Malaria, HIV Type 1
• Advantages of Yeast - Protein Engineering

  • Yeast are generally safe, inexpensive and easy to observe
  • The genomes of the commonly used yeast have been sequenced and genes have been mapped making it a useful tool for biopharmaceutical industry
  • Yeast can synthesise more complex eukaryotic proteins
  • The yeast can make the required modifications to the proteins after being translated
  • They grow and reproduce quicky, therefore produce large quantities of the protein quickly too
• Limitations of Yeast - Protein Engineering

  • Yeast can produce methanol which is flammable
  • Glycosylation (addition of carbohydrates) of proteins is different in yeasts compared to in human cells, which can lead to less effective vaccines
• Steps of DNA Cloning

  1. Extract DNA from target cell containing the gene of interest
  2. Cut the target DNA using a specific restriction enzyme (this produces multiple DNA fragments with sticky ends containing unpaired bases)
  3. Remove plasmids from bacteria
  4. Cut the plasmids with the same restriction enzyme (this produces complementary sticky ends)
  5. The DNA fragments are inserted into the bacterial plasmids and annealed using DNA ligase (forming recombinant plasmids, one of which contains the gene of interest)
• Steps of DNA Cloning (2)

  1. Bacterial cells take up recombinant plasmids in a process called transformation. This involves shocking them using electricity (electroporation) or high temperatures
  2. All transgenic bacteria are then cultured (DNA replication and binary fission occurs leads to many copies of modified bacteria quickly)
  3. This exponential growth of bacteria results in multiple copies of the gene of interest being produced (e.i. DNA cloning)
• Steps of DNA Cloning (3)

  1. The transformed bacterial colony containing the gene of interest is identified using a specific fluorescent DNA probe that is complementary to a section of the gene of interest
  2. The fluorescent colony that the probe binds to is isolated from the other colonies and is cultured separately
  3. If the goal is to produce and harvest a human protein (e.g. insulin) the protein is harvested and processed for human use
• Steps of DNA Cloning (4)

  1. However, if the goal is to transfer the gene of interest into another organism, the multiple copies may need to be isolated for transfer (depending on the method). This is applicable to both transgenesis and gene therapy
• Transgenesis

  • An organism whose genome has been altered by the introduction of one or more foreign DNA sequences from another species by artificial means
  • Can occur physically, chemically, or using a vector such as bacteria or viruses
• Microinjection

  • Physical method of gene transfer.
  • Transfers genes to animal cells.
  • Many copies of the desired gene are injected into the nucleus of a fertilised egg cell.
  • A thin micropipette is used.
  • Requires highly skilled technicians.
  • Also used in IVF (in vitro fertilisation).
• Viral Vectors

  • Viruses genome can be edited.
  • Virus is inserted into target cell.
  • Desired gene is inserted in the cell’s genome.
• Electroporation

  • Electric pulse is used to “break” the cell membrane.
• Ti Plasmid

  • Bacteria that copies plants Ti (tumour inducing) plasmid. Transfers copy to plant cell. Scientists replace this with desirable gene.
• Genetically Modified Organisms (GMOs) - Examples

  • Golden Rice/Bananas – added vitamin A
  • Herbicide resistant crops – used to kill weeds and keep crops
  • Pest resistance crops – used to kill insects (pierces holes in gut cells)
  • Disease resistant crops – insert desirable genes that prevent disease into crops (e.g. cavendish bananas and disease)
  • Increased shelf life – tomatoes which prevent enzyme from breaking down cell wall and softening tomato (similar to pectin/pectinase)
• Genetically Modified Organisms (GMOs) - Examples

  • Used to make fluorescent fish - boost pet industry
  • Florescent markers are used for cancer treatment
• CRISPR/Cas9

  • Viruses naturally infect bacteria, killing them.
  • Bacteria have a defence mechanism called CRISPR.
  • (1)Virus infects bacteria.
  • (2)Copy of viral DNA is stored in bacteria DNA called CRISPR.
  • (3)CRISPR sequence allows bacteria to remember previous viral invaders.
  • (4)If reinfected with virus, guide RNA and Cas enzymes are sent to kill the virus.
  • This has been modified by scientists to edit DNA by removing, adding or altering sections of DNA.
• Two key molecules with CRISPR/Cas9

  An enzyme called Cas9

  • Cas9 acts as a pair of 'molecular scissors' that can cut the double stranded DNA of hose cell at a specific location

  Guide RNA (gRNA)

  • A short RNA sequence (about 20 bases long) that is synthetically made to be complementary to a specific section of DNA. The guide RNA binds to DNA and 'guides' Cas9 to the right part of the genome to ensure that the Cas9 enzyme cuts at the right point in the genome
• CRISPR/Cas9

  • Cas9 can be programmed using guide RNA to cut genes at specific points.
  • need to know specific target DNA sequence to synthesise guide RNA that is complementary to desired gene sequence (use of DNA sequencing).
  • Guide RNA and Cas9 protein are transferred into target organism.
  • Cas9/guide RNA complex scans DNA in organism.
  • Finds complementary section. Guide RNA guides Cas9 enzyme to cutting site, breaking both strands.
• CRISPR/Cas9 - Applications

  • CRISPR/Cas9 is currently the fastest and best system for gene editing.
  • Can be applied to any species.
  • Research of this technology continues to advance.
  • Treating HIV, muscular dystrophy, sickle cell disease, cystic fibrosis, Alzheimer’s disease.
  • Scientists have made a version that can turn specific genes on and off (gene expression) without cutting the DNA.
  • This can alleviate ethical concerns around permanent DNA changes.
• CRISPR/Cas9 - Applications

  Cure genetic diseases

  • Potential to cure genetic diseases such as sickle cell anaemia, cystic fibrosis, and Duchenne muscular dystrophy (DMD)

  Designer babies and pets

  • Parents could potentially choose desired traits and 'design' their baby or pet using this technology.

  Agriculture

  • Creating crops that are disease resistant, non-browning, drought tolerant, etc. to produce higher yields
• Limitations of CRISPR/Gene Editing

  Off-target activity

  • There is a potential for CRISPR/Cas9 to cut in the wrong location if the guide RNA sequence is also located in other areas of the genetic code. A possible consequence is cancer

  Mosaic Generation

  • Treated patients could have a mixture of treated and untreated cells

  Immune system Complications

  • The CRISPR/Cas9 treatment could trigger an immune response. The immune system could see Cas9 as a foreign protein and initiate inflammatory response
• Limitations of CRISPR/Gene Editing

  Research on germ line cells

  • Because any changes made in germline cells will be passed on from generation to generation, it has important ethical implications that must be considered (e.g. the infant produced from these cells cannot consent to the treatment)

  Illegal research

  • The technology is cheap, easy to perform and readily available raising concern of unauthorised research. An example of this has occurred recently, where a scientist performed secret experiments on twin embryos using CRISPR to edit a gene associated with resistance to HIV.
• Social and ethical issues

  • Bioethics involves the study of ethical issues in biology.
  • Deals with basic human rights and values.
  • Disciplines include law, philosophy, theology and social science.
  • How does society keep pace with laws etc. when technology moves so quickly? (think of current issue with AI)
  • Issues fit in categories including ethical, social, environmental, economic, individual, cultural and religious.
• Social and Ethical Issues

  Some examples include:

  • Unknown side effects/long term effects
  • Pest resistant genes transferring to weeds, making them dominant to native species.
  • Who owns the GMO? Private companies profiting off consumers.
  • Exploiting of vulnerable individuals.
  • Essentially, who are we to play God?
• Designing New Proteins

  • As we know, the sequence of amino acids determines the secondary and tertiary structure of proteins, and it’s function.
  • Protein folding problem – how the proteins amino acid sequence determines the 3D shape.
  • If scientists solve this problem, this will help with designing new proteins from scratch – would make other methods redundant.
  • If scientists can design proteins on the computer, they’ll be able to synthesise actual proteins (using DNA strand) using biotechnology.
  • AI system called AlphaFold is already being used.
• Designing New Proteins

  • Discovering and engineering enzymes to digest plastic waste
  • Creating new and more efficient vaccines for diseases such as SARS-CoV 2 and RSV
  • Creation of narrow spectrum drugs that focus on one target reducing side effects
  • Creation of new synthetic antibiotics to combat antibiotic resistance and 'superbugs'
  • Development of new materials such as protein sheets that could be used in solar cells
  • The creation of new proteins for cancer immunotherapy
• Describe the main step requires to genetically manipulate cotton plants to be resistant to a specific herbicide

  A donor species with a gene resistant to the herbicide needs to be identified. The gene is removed, placed in a donor and transmitted to the cotton plant which can then be cloned as a GMO
• Describe on benefit of genetically manipulation cotton plants to be resistant to a specific herbicide

  It would enable farmers to spray the chemical directly on the cotton crop to kill weeds without impacting on the cotton plant itself, increasing yield and profits
• Name three specific proteins, or classes of proteins, made using biotechnology and state an application of each

  Hormones

  • Insulin is used to treat people suffering from diabetes

  Enzymes

  • Cas9 is a restriction enzyme used in cutting genes from DNA

  Antigens

  • Used in vaccinations to confer immunity to specific diseases

  Antibodies

  • Used to provide passive immunity to specific pathogens
• Describe two reasons why some people may have concerns about genetically modified food

  • Individuals or groups may have religious beliefs that centre around not altering organisms in any way
  • They may not have an understanding of DNA and genetic engineering and therefore be hesitant to consume something they don't understand
  • They could have heard misinformation from friends or social media resulting in unwarranted concern
  • They may have concerns about possible unknown side effects of such food
• Describe two possible negative impacts of genetically modified crops on ecosystems

  • Genes that have been transferred to a 'target species' ma also accidentally be transferred to unintended species through cross-contamination and have unintended results
  • Such crops may disrupt the food webs by changing balance and interactions between species currently in equilibrium
  • Weeds may become resistant to herbicides used and more toxic ones may be requires, which could contaminate waterways and higher order species through biomagnification
• Some proteins in fruits and vegetables are involved in ripening. Delayed ripening GM FlavR SavR tomatoes were one of the first examples of genetically modified foods approved for sale to consumers. Suggest how the addition of a gene to these GM tomatoes can prevent tomatoes from ripening early.

  Tomatoes can ripen early because of specific proteins associated with ripening. If the production or availability of one of these ripening proteins was disrupted, early ripening would be prevented. Disrupting ripening could be inserting genes that code for proteins that degrade ripening proteins.
• State the function of the protein 'Cas 9' in the process

  This is similar to restriction (endonuclease) enzymes, Cas9 cuts DNA at particular sites
• Describe how guide RNA works in ensuring that the action of 'Cas 9' occurs in the right position

  Guide mRNA would bind in a complementary manner to the DNA adjacent to the cutting site
• Describe how the guide RNA might end up binding in an undesired position

  It may bind to a complementary section but not adjacent the specific gene that was needed because there could be multiple repeats of the code in the species' genome
• Genetic manipulation and the production of transgenic organisms pose a range of potential issues. Describe two possible social and/or ethical issues

  • It is a matter of personal choice, particularly if it involved the mistreatment of animals
  • There are issues surrounding accessibility to the technology, particularly if it is for commercial gain by individuals or businesses
  • There is the possibility that we cannot safeguard against exploration for commercial gain and unethical use
• Describe two reasons in favour of exercising that biotechnology enables parents to be able to select the gender of a child

  • Medical reasons - some genetic diseases are linked to gender
  • Family reasons - some may have several children of one gender and desire the other gender