Genetic engineering Flashcards
What are restriction enzymes/endonuclease?
Enzymes that cut along specific nucleotide sequences (restriction sites) to leave either blunt ends or sticky ends by catalysing a hydrolysis reaction that breaks sugar-phosphate backbone along the particular sequence.
What are the characteristics of a restriction site?
- Less than 10 bp long.
- Enzyme cuts DNA in restriction site in a staggered manner.
- The complementary base sequence in restriction site are palindromic, in the sense that one is the backwards version of the other.
- The cut in the backbone by the enzyme is usually made between the same 2 nucleotides on both strands.
How can a desired gene be obtained?
- Using restriction endonuclease to cut out a desired gene from the genome of an organism (leaving sticky ends). Use DNA probes and electrophoresis to locate and isolate the desired gene.
- Isolate mRNA from a cell. Use reverse transcriptase and DNA polymerase to synthesise a complementary DNA (cDNA) gene.
- Sequence a desired protein to work out the required gene and build the gene from scratch.
What different types of vectors are there?
- Virus cell genome: Gene inserted into host cell when infected by virus.
- Yeast cell chromosomes.
- Bacterial plasmids: Inserted directly into bacteria (most common approach).
- Liposomes: Artificial vesicles that enclose the desired gene and insets it into host cell by fusing with plasma membrane.
- Bacteria Artificial Chromosomes (BACs).
- Ti Plasmids: Inserted into soil bacteria Agrobacterium tumefaciens which infects plants and inserts plasmids into plant genome.
How is desired gene inserted into a bacterial plasmid?
- Cut plasmids open with same restriction enzyme used to cut out gene initially, leaving same complementary sticky ends as on gene.
- Mix gene with open plasmids and DNA ligase, which joins complementary sticky ends on gene to the ones on plasmid by catalysing condensation reaction which reforms sugar-phosphate backbone, as helping form hydrogen bonds between complementary nucleotides.
- Gene is sealed into the bacterial plasmid to form recombinant plasmid.
What is recombinant DNA?
DNA which is formed by joining DNA from 2 different organisms.
How is bacterial plasmid put into host cell?
- Electroporation: The host cells are given a high-voltage electric shock, which disrupts plasma membrane and makes it more permeable for short amount of time; enough for vector to be taken up.
- A very fine micropipette is used to directly inject recombinant DNA into host cell.
- Calcium salt is added to mixture of host cell and large quantities of vector and heat shock is carried out; whereby temperature is dropped to near freezing and quickly raised to 40ºC.
How are bacteria that have taken up the vector plasmids identified?
The vector plasmid usually has a gene for antibiotic (e.g. tetracycline) resistance. When bacteria are grown on agar containing tetracycline, only transgenic bacteria that have taken up plasmids live (since they are resistant). The ones that haven’t are killed.
How are bacteria that have taken up the plasmids with desired gene identified?
The vector plasmid usually contains 2 genes coding for resistance to 2 different antibiotics (e.g. tetracycline and ampicillin). Restriction site where plasmid is opened and gene inserted is usually in the middle of 1 of the 2 antibiotic resistance genes (e.g. ampicillin). Insertion of desired gene into antibiotic resistance gene deactivates resistance gene. Bacteria that take up just vector plasmid are resistant to both tetracycline and ampicillin while one that have taken up plasmid with desired gene are only resistant to tetracycline.
Method:
1. Grow bacteria on standard agar dish.
2. Use velvet pad to transfer colonies onto agar dish containing ampicillin and another containing tetracycline.
3. Compare colonies on both dishes. The ones that have disappeared on ampicillin dish are ones that have taken up desired gene.
4. Isolate identified colonies and culture them.
This technique is called velvet plating.
What are the advantage of bacteria taking up new plasmids?
- Conjugation, the process of different bacteria exchanging genetic information in the form of plasmids.
- This allows them to pass on desired characteristics such as antibiotic resistance, which increases their speed of transfer across population and increases their chance of survival.
How are transgenic bacteria that produce human insulin created?
- mRNA coding for insulin isolated from β-cells by centrifugation.
- Reverse transcriptase and DNA nucleotides added to mRNA, which synthesises a single strand of cDNA; complementary to that of the original coding strand.
- DNA polymerase added to mixture, which uses cDNA strand as template to build original coding strand onto template strand to form a copy of original double stranded DNA coding for insulin gene (called cDNA gene).
- Restriction enzymes added to cut ends of insulin gene to form sticky ends.
- Bacterial plasmids are cut open with same restriction enzyme to form complementary sticky ends to those on gene.
- Insulin gene mixed with open plasmids and DNA ligase, which joins gene sticky ends to open plasmid sticky ends, inserting gene into plasmid.
- Recombinant plasmid introduced into bacteria using range of techniques (e.g. heat shock).
- Various techniques are used to identify transgenic bacteria containing insulin gene.
- Transgenic bacteria are then cultured to extract insulin produced.
What are the steps involved in producing transgenic golden rice?
- Gene coding for phytoene synthase extracted from genome of daffodils using restriction enzymes.
- Gene coding for Crt 1 enzyme extracted from genome of bacteria called Erwinia uredovora, also using restriction enzymes.
- Bacterial plasmid cut open with same restriction enzymes as those used to extract previous 2 genes.
- Genes and promoters were mixed with open plasmids along with DNA ligase, allowing them to be inserted into plasmid.
- Recombinant plasmids were then inserted into bacteria called Agrobacterium tumefaciens.
- Transgenic Agrobacterium mixed with rice embryos in petri dish, which allowed them to infect rice embryos, inserting the genes and promoters into the genome of some.
- The transgenic embryos can then be grown to into adult plants that produced endosperms containing β-carotene, giving them the distinct ‘golden’ colour.
Why is β-carotene consumption important in balanced diet?
- β-carotene is a precursor molecule to vitamin A, which can be produced inside body if not enough is consumed.
- Many people in developing countries don’t have balanced diets or consume enough vitamin A.
- They become vitamin A deficient and their bodies have no ways of producing it.
- Vitamin A deficiency may potentially result in blindness.
Why do only 2 genes need to be inserted into rice for it to produce β-carotene?
Because the rice already contained most of the enzymes required in the metabolic pathway of β-carotene synthesis.
What is gene therapy?
Therapeutic technique involving placing a functional allele or a particular gene into the cells of individuals lacking that functional allele. This means it can only be used to treat recessive conditions.
