Core R - Gene Tech Flashcards
Explain why bacteria are first spread onto plates containing (drug name) [first plate in replica plating]. (3)
- Identifying bacteria that are transformed (have taken up plasmid that has the resistance gene for the drug)
- These bacteria have survived
- These bacteria may contain the recombinant plasmid
- Other bacteria have been killed
Describe how a cDNA human insulin gene can be inserted into a plasmid that has been cut by a restriction enzyme. (4)
- Sticky ends
- (refer to diagram)
- Complementary bases pairing
- A-T and C-G
- H bonds to sticky ends of plasmid
- Gaps in sugar-phosphate backbones sealed by DNA ligase
- Formation of phosphodiester bonds
Explain why it is important, for identifying bacteria that have successfully taken up the recombinant plasmid, that on the original plasmid the target site for the rest. enzyme is in the middle of the second resistance gene. (3)
- Enzyme breaks the resistance gene
- Inserting human insulin gene makes the resistance gene inactive
- Colonies that are resistant to first drug but not to second, have taken up recombinant plasmid (insulin gene)
- Colonies that survive on first and second antibiotic have not taken up the recombinant plasmid
Explain why antibiotic resistance genes are now rarely used. (2)
- Plasmids are easily transferred between bacteria
- Of the Same species or between different species
- Bacteria can acquire antibiotic resistance (renders antibiotic useless)
State one type of gene that has replaced antibiotic resistance genes in plasmid vectors and indicate how its presence can be detected. (2)
- Gene for B galactosidase ; blue colour from X-gal medium
- Gene for B glucuronidase (GUS) ; produces product that is easily stained blue
- Gene for GFP ; fluorescence detected when present
Outline how a gene from another species can be inserted into E. coli. (3)
- Gene isolated
- Inserted into plasmid
- Sticky ends
- Plasmid taken up by E. coli
- Use of restriction enzyme
- cDNA produced
Explain how a marker gene, such as the gene for GFP, is used to show successful uptake of a gene for a wanted protein. (3)
- Marker gene linked to gene for wanted protein
- With promoter
- GFP gene is transcribed
- Producing GFP which fluoresces
Suggest one disadvantage of using the gene for GFP (not an enzyme) to produce easily detectable fluorescence, rather than using a gene for an enzyme that produces a fluorescent substance.
• May not fluoresce very brightly (difficult to detect)
Because:
- Only a few molecules of GFP produced
- Each enzyme molecule produces more fluorescent substance (enzymes can be re-used)
Steps in a method of production of human insulin by gene technology.
- mRNA for human insulin isolated in B-cells
- cDNA coding for human insulin synthesised
- DNA for human insulin cloned
- DNA coding for human insulin inserted into cut plasmid vector
- Plasmid vector inserted into bacterium
- Genetically modified bacteria identified
- Genetically modified bacteria cloned
- Human insulin harvested
Name the enzymes responsible for:
- Insertion of human insulin into cut plasmid vector
- Synthesis of cDNA coding for human insulin
- DNA ligase
* Reverse transcriptase
Explain two advantages of treating diabetes with human insulin produced by gene technology rather than using insulin from animals. (2)
- Identical to human insulin
- More rapid response
- No rejection problems / side effects / allergic reactions
- Ethical / moral / religious issues
- Cheaper to produce in large volume / unlimited availability
- Less risk of transmitting disease / infection
- Good for people who have developed tolerance to animal insulin
State the reasons for the steps below:
- Obtain copies of gene with sticky ends
- Plasmid used
- Use restriction endonuclease enzyme
- Mix vector and gene
- Use DNA ligase
- Insert plasmid / vector into host / bacteria
- Screen for and obtain successfully transformed cells
- Fermenter
- Gene codes for the synthesis of insulin
- Acts as a vector for the transfer of the gene into the host
- To produce sticky ends (cut a specific site / sequence)
- Gene inserts into vector / plasmid (forms recombinant DNA)
- To seal sugar-phosphate backbone
- To obtain transformed E. coli cells
- So only recombinant host cells cultured
- To obtain large amounts of insulin for extraction and purification
Suggest why isolating the mRNA coding for insulin in a B-cell is easier than isolating the DNA for insulin in B-cell. (2)
- mRNA is only from gene coding for insulin
- Large number of mRNA coding for insulin
- DNA has all genes
- Must use restriction enzymes
Outline the use of restriction enzymes in preparing plasmid vectors. (2)
- To cut the plasmid DNA
- At a specific site / base sequence
- To produce sticky ends that will join with the insulin gene
Describe the advantages of treating diabetics with insulin produced by gene technology. (3)
- Identical to human insulin (Fits membrane receptor on target cells)
- More rapid response
- No rejection problems / side-effects / allergic reactions
- Ethical / moral / religious issues
- Cheaper to produce in large volume / unlimited availability
- Less risk of transmitting disease / infection
- Good for people who have developed tolerance to animal insulin
