In Vivo Gene Cloning
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What does 'in vivo' mean?
Using living cells
Why are microbes often the recipients of foreign genes?
- They reproduce rapidly
- They can produce many cloned copies of a gene
How do Bacterial cells store genetic information?
Within plasmids, small circles of DNA
How are plasmids utilised by geneticists?
They can be easily extracted from the bacterial cell and can be used as vectors to carry genes into the bacterium
What is a genetically engineered plasmid?
A plasmid with a foreign gene inserted into it
How can GEPs be identified?
- Some have natural genes that provide antibiotic resistance
- This is used as a genetic marker with bacteria grown on agar containing antibiotics
- The bacteria containing the plasmid and the resistance will survive
Describe stage 1 of foreign gene isolation into a bacterium
The DNA containing the foreign gene is isolated from a cell
Describe stage 2 of foreign gene isolation into a bacterium
A foreign gene is cut out of the gene using restriction endonuclease
Describe stage 3 of foreign gene isolation into a bacterium
- Plasmid DNA is cut at a single site using the same restriction endonuclease to linearise the plasmid
- The plasmid will contain 2 marker genes
- The recognition sequence for the restriction endonuclease will be in the middle of the second gene
Describe stage 4 of foreign gene isolation into a bacterium
- The foreign gene and the plasmid DNA are mixed with the sticky ends joining due to hydrogen bonding between complementary bases
- Most of the plasmids recircularise without the foreign gene being inserted
Describe stage 5 of foreign gene isolation into a bacterium
The foreign gene will deactivate the gene for antibiotic resistance (such as tetracycline)
Describe stage 6 of foreign gene isolation into a bacterium
A ligase enzyme joins the two fragments together forming phosphodiester bonds between the sugar and the phosphate
Describe stage 7 of foreign gene isolation into a bacterium
- The plasmid is reinserted into bacterial cells
- This process is known as transformation
- The bacteria are left to recover for a while in order to allow them to express their antibiotic resistant genes
Describe stage 8 of foreign gene isolation into a bacterium
- To select the bacteria containing the plasmid the bacteria is grown on agar containing ampicillin
- Only bacteria containing the plasmid will grow as the ampicillin resistance gene is present
- Those not containing the plasmids will die
Describe stage 9 of foreign gene isolation into a bacterium
- When bacterial colonies have formed a sterile velvet cloth is laid atop the medium allowing a few bacterial cells from each colony to be picked up
- The bacterial cells are then transferred to another plate containing the tetracycline
- This method is known as replica plating and should transfer all of the colonies to another plate in exactly the same position
Describe stage 10 of foreign gene isolation into a bacterium
- Bacteria that grow on the plate containing ampicillin but not on the plate containing tetracycline must contain plasmids with the foreign gene inserted into them
- This is because they will have the active ampicillin resistance gene but the tetracycline resistance gene will be inactivated by the insertion of the foreign gene
Describe stage 11 of foreign gene isolation into a bacterium
The bacteria can be selected by picking the colony on the ampicillin plate that was not present on the tetracycline plate
How can bacteria containing foreign genes be grown on a large scale?
By using industrial fermenters
What conditions must be met to ensure a high rate of cell division?
- High concentrations of glucose and oxygen
How can proteins be yielded from bacteria colonies?
The protein is expressed by the bacteria and can be extracted from the growth medium
Give one issue with this method of gene cloning?
Antibiotic resistant strains of bacteria could transfer their resistance genes to pathogens via conjugation
Give one example of an alternate genetic marker
- The use of plasmids with a gene that produces a green fluorescent protein
- Insertion of a foreign gene in the middle of the GFP gene will inactivate it and so bacteria containing recombinant plasmids can be identified as they will be resistant to ampicillin but will not fluoresce
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