Outcome 1 Flashcards

Question

Steps in the Production of Human Insulin Using Recombinant Plasmids

Answer 1

1. Isolating the Human Insulin Gene - Scientists extract the human insulin gene from a DNA sample. - A restriction enzyme cuts the DNA at specific sites, creating sticky ends. 2. Preparing the Plasmid Vector - A plasmid (a small, circular DNA molecule found in bacteria) is used as a vector to carry the insulin gene. - The same restriction enzyme cuts the plasmid, creating complementary sticky ends. 3. Inserting the Insulin Gene into the Plasmid - The insulin gene is inserted into the plasmid. - DNA ligase seals the human gene into the plasmid, forming a recombinant plasmid (a plasmid containing foreign DNA). 4. Transforming Bacteria with the Recombinant Plasmid - Bacteria are treated with the recombinant plasmid using heat shock or electroporation, allowing them to take up the plasmid. - The transformed bacteria now have the human insulin gene. 5. Selecting Transformed Bacteria Not all bacteria successfully take in the plasmid, so an antibiotic resistance gene is included in the plasmid. Bacteria are grown on an antibiotic-containing agar plate: Bacteria with the plasmid survive (they carry the resistance gene). Bacteria without the plasmid die. This ensures that only transformed bacteria are selected. 6. Producing and Harvesting Human Insulin The transformed bacteria express the human insulin gene, producing insulin protein. The insulin is extracted, purified, and used for diabetes treatment.

Answer 2

✅ Efficient and Large-Scale Production: Bacteria can rapidly multiply, producing large amounts of insulin. ✅ Safer than Animal Insulin: Previously, insulin was extracted from animals (e.g., pigs), which could cause allergic reactions. ✅ Genetically Identical to Human Insulin: Reduces the risk of immune system rejection. ✅ high levels of purity ✅ reliability of supply ✅ consistency of quality between batches.

Answer 3

The DNA is cut at one point using restriction enzymes – sticky ends. The DNA passenger fragments are prepared using the same restriction enzyme. The passenger fragments are mixed and their ‘sticky ends’ pair. The joining enzyme, ligase, makes the joins permanent. Plasmids that contain the DNA passenger are then selected from the mixture. These plasmids with their passenger DNA can be reintroduced to bacterial cells using an electric pulse or heat shock.

Answer 4

Once recombinant plasmids have been created, plasmids need to be transferred into bacterial cells. This process is known as bacterial transformation. The bacterium needs to be able to reproduce quickly and be harmless to individuals working with it. A harmless strain of E. coli is commonly used for this purpose.

Answer 5

Cells are briefly placed in an electric field Cells are shocked “Holes” are created in their plasma membranes Plasmid entry is facilitated.

Answer 6

Step 1: Produce the two insulin chains separately. (As the amino acid sequence is known for each of these chains, this can be used to determine the DNA sequence that will lead to the production of insulin). Step 2: The DNA is often produced using reverse transcription. Mature mRNA is used as it no longer contains non-coding introns. With the aid of the reverse transcriptase enzyme, the DNA segments coding for insulin can be produced. Step 3: The insulin gene and plasmid are cut with an endonuclease (usually BamHI), forming sticky ends. The two DNA molecules are then inserted into plasmids using ligase. Step 4: The plasmids are then inserted into the bacterium E. coli. The plasmid contains the selectable marker the lacZ gene. LacZ encodes for β -galactosidase. Step 5: The recombinant plasmids are then mixed with the bacterial cells. Plasmids enter the bacteria in a process called transformation (using either heat shock or electroporation). The bacteria culture is incubated, allowing for the replication of bacteria and plasmids. Step 6: Bacteria are spread over culture plates containing ampicillin and X-gal. Bacteria with the recombinant plasmid are able to grow in the presence of ampicillin. They CANNOT express beta galactosidase, hence cannot digest X-gal. Colonies are therefore WHITE in colour. Step 7: The bacteria with the recombinant plasmid undergo a fermentation process. The millions of bacteria replicate roughly every 20 minutes, expressing the insulin gene. Step 8: Cells are then lysed using lysosomes, where the expressed protein is able to be extracted. The protein is then treated with cyanogen bromide, separating the insulin chains from other proteins that were also translated. Step 9: The purified samples can be tested to ensure none of the bacteria's E. coli proteins are mixed in with the insulin. This is done by using a marker protein to detect the E. coli DNA Step 10: The two chains are then mixed together and re-joined by forming disulfide bonds (or bridges). Step 11: At the end of the manufacturing process, ingredients are also added to intermediate and long-acting insulin to produce the desired duration type of insulin

Answer 7

Genetically Modified Organisms (GMOs): Organisms whose DNA has been altered using genetic engineering. Transgenic Organisms (TGOs): A subtype of GMOs that contain genes from a different species. - GMO = If the gene is from the same species - GMO & TGO = If the gene comes from a different species All TGOs are GMOs, but not all GMOs are transgenic.

Answer 8

Herbicide-resistant crops (e.g., Roundup Ready soybeans) Contain a gene that makes them resistant to glyphosate, allowing farmers to kill weeds without harming crops. Pest-resistant crops (e.g., Bt cotton & Bt corn) Produce a natural bacterial toxin (Bt toxin) that kills insect pests. Reduces the need for chemical insecticides. Disease-resistant crops Some GM bananas are resistant to Panama disease, a fungal infection that threatens global banana production.

Answer 9

Cells are suspended in an ice-cold salt solution (~30min) Then transferred to 42 °C (<1min) Increases the fluidity of the cells’ plasma membranes Increases the chance of uptake of plasmids

Answer 10

Group 1: Herbicide-resistant crops (glyphosate) - HERBICIDES kill weeds (or any plant material) - These crops were first altered so that they were resistant not affected by the herbicide glyphosate (active ingredient in the commercially purchased herbicide Roundup). farmers could eliminate weeds without harming their crops. This increased crop efficiency while eliminating weeds. Group 2: Natural pesticides and insecticides - These crops are altered to produce their own natural pesticides. - Protect the crops against insect infestation, as the pesticide is located within the plant and therefore needs to be ingested by the insects to have an effect. - The use of natural pesticides is environmentally friendly as it eliminates the use of sprays that could be harmful and toxic to other organisms.

Answer 11

GENETIC TECH: insertion of genes into plants so they gain desirable features such as disease resistance, firmer fruits or resistance to herbicides ETHICAL CONSID: → Environmental: it is possible that genes from Gm crop plants will cross into non-GM varties via pollen or seeds it will also spread through bacterial or viral infections → evolution: greater pressure for farmers to use higher- yielding or more resistant crop varieties, thus reducing variety. → ethics: - is it right that cooperation's hold ownerships over crops that have been grown for centuries, just bc they developed one genetic variety ? - are these varieties squeezing out traditional farming in poorer communities

Answer 12

1. SOCIAL ISSUES Social issues affect society. For example, are person who is unable to work due to a genetic issue. Socially, if genetic testing were available, the individual would have less reliance on hospitals in the future. This affects more than the person therefore is a social issue. If we just stated that the person would be unwell, this affects the individual but not society as a whole. 2. LEGAL ISSUES Legal issues are often governed by legislation and government regulation. As ethical issues are often using new technologies, the laws may not be current. 3. ECONOMIC ISSUES Economic factors include areas such as bioprospecting and who owns the rights to materials. Is it the company who conducted the research or is it the locals that reside in an area? Who owns a genome, and is it fair to allow large companies to grow crops that are GM and more likely to survive, when the sole farmer

Answer 13

DUTYAND/OR RULE BASED decision is aligned with a set of rules with the consequences not considered in the decision making process PROS: simple to apply CONS: rigid application VITURE BASED APPROACH: moral character of the person is considered. PROS: not binding on all people CONS: a virtue is subjective CONSEQUNCES BASED APPROACH: the outcome of the decision is considered with minimization of harm a key consideration PROS: flexible decision making CONS: hard to predict outcome

Answer 14

Integrity (HONEST): the commitment to searching for knowledge and understanding and the honest reporting of all sources of information and communication of results, whether favourable or unfavourable, in ways that permit scrutiny and contribute to public knowledge and understanding. Justice ( FAIR ): the moral obligation to ensure that there is fair consideration of competing claims; that there is no unfair burden on a particular group from an action; and that there is fair distribution and access to the benefits of an action. Beneficence (BENEFIT): the commitment to maximising benefits and minimising the risks and harms involved in taking a particular position or course of action. For example - have considerations been taken to ensure that the planting of a new crop will benefit the farmer, the consumer, and the ecosystem ring harm Non-maleficence ( NO HARM ): involves avoiding the causations of harm. However, as positions or courses of actions in scientific research may involve some degree of harm, the concept of non-maleficence implies that the harm resulting from any position or course of action should not be disproportionate to the benefits from any position or course of action. Respect (VALUING): involves consideration of the extent to which living things have an intrinsic value and/or instrumental value

Answer 15

The leader sequence (trpL) has a small section of mRNA that the ribosome starts translating. This sequence contains two tryptophan codons (UGG-UGG). If tryptophan is abundant, the ribosome moves quickly The ribosome blocks part of the mRNA (region 2), forcing regions 3 and 4 to pair. This forms a terminator hairpin (a loop that tells RNA polymerase to stop). Transcription stops before the genes needed for tryptophan synthesis are made

Outcome 1 Flashcards

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