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Principles of Genetic engineering
To describe the main stages in genetic engineering
Genetic engineering: recombinant DNA technology,
– altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype.
• inserting a foreign gene from one species into another, forming a transgenic organism
• altering an existing gene so that its product is changed
• changing gene expression so that it is translated more often or not at all.
Purpose of GMOs
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Improved feature
–
Herbicide resistance gene inserted into a plants’ genome
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For synthesis of useful products
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For synthesis of useful products
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Human hormones mass-produced in bacteria
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insulin, growth hormone
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-carotene in rice grains
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turns into vitamin A when eaten
Basic steps in genetic engineering
1. Isolate the gene
2. Insert into a vector
3. Insert modified vector into host cell (Transformation & selection)
4. Allow host to multiply and to synthesise protein
5. Separate and purify the product of the gene
Step 1: Isolating the gene
A. Using restriction enzymes
1. A probe is used to locate the gene
2. Restriction enzymes recognise restriction sites and cut
out gene
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Restriction sites are palindromic
– The same sequence when read left to right (5’ to 3’) on one strand and right to left on complementary strand
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Originally obtained from bacteria
– Cuts up phage DNA
– DNA of virus that infect bacteria
– Why is this useful?
– Why are the staggered cuts called sticky ends?
3. Terminal transferase enzyme can add sticky ends if restriction enzyme generates blunt ends
B.
Using Reverse transcriptase
• Gene for this enzyme originally found in retroviruses (contain RNA instead of DNA)
• why is the enzyme useful for these?
• Converts mRNA into single-stranded cDNA
– E.g. insulin mRNA from B-cells of islets of Langerhans
• Then DNA polymerase produces complementary strand to form double stranded DNA
• Advantage – more mRNA in cell than DNA
• Why is it an advantage to use cDNA if you are inserting a eukaryotic gene into a prokaryote?
C.
Using an automated sequencer
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Amino acid sequence of protein analysed
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Gene for protein synthesised
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Using triplet code
Step 2: Inserting gene into a vector
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Vector – molecule of DNA which is used to carry a foreign gene into a host cell, e.g.
– Bacterial plasmids
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double stranded circular DNA
– Virus genomes
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Can carry large fragments
– Yeast cell chromosomes

•
•
Recombinant DNA
DNA from different sources
That have been combined
Plasmid and foreign DNA are cut with the same enzyme
– Generates complementary sticky ends that can anneal
Ligase enzyme seals gaps/nicks in S-P backbone
– forms phosphodiester bonds between inserted gene and the
plasmid
Step 3: Transformation (bacteria taking up plasmid)
1. Soak E.coli in CaCl, mix with plasmid, mild heat shock
–
Makes membrane more permeable to plasmids
2. Electroporation
– High voltage pulse disrupts membrane
3. Microinjection
– using fine micropipette
4. Viral infection
– using virus’ own mechanism to insert DNA
5. Bacterial infection
– Agrobacterium tumefaciens which naturally insert Ti
plasmids into plant genome
6. Liposomes
– containing DNA easily cross lipid membrane
Selection
Colonies of bacteria will grow from each bacterial cell which
–
Did not take up plasmid
–
Took up plasmid which re-sealed without inserted gene
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Transformed bacteria : Those that took up recombinant plasmids (i.e. have inserted gene)
How can we tell the difference?

Using antibodies for protein produced

Adding a fluorescent marker gene to plasmid
o Glowing bacteria have plasmid

Replica plating
Replica plating

Using plasmids with 2 genes for antibiotic resistance

Plasmid used carries
– ampicillin resistance gene (amp)

Allows only transformed bacteria to grow in agar plates with ampicillin antibiotic (‘amp plates)’
– Tetracyclin resistance gene (tet)

Gene is inserted within this gene
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Tet gene inactivated

Transformed bacteria cannot grow in ‘Tet plates’

Those that grow in amp plates but not in tet plates are transformed bacteria
Step 4: Multiplication of the host cells by cloning

Large scale fermenters

Bacteria undergo binary fission
o Large numbers produced quickly
o E. coli divide every 20 min
o All genetically identical because of asexual reproduction

Transcription and translation
Step 5: Extraction and purification of protein

Bacteria killed and separated from proteins by centrifugation

Protein of interest separated from others by
– Large scale chromatography
– ultrafiltration