Transcript 11. Genetic engineering case study 1 - Human Insulin

03 April 2016
Today’s Title: CW: Genetic engineering case studies –
1 human insulin
Learning Question: how can insulin be manufactured?
Aims from specification
(n) outline the process involved in the
genetic engineering of bacteria to produce
human insulin;
Reverse transcriptase
• Enzyme that retroviruses carry to convert their
RNA into DNA – the reverse of transcription
• Scientists have used this to build copies of
human genes from isolated mRNA
• Single-stranded copy of DNA is formed from
mRNA
Genetically engineering insulin
• Stages to isolate the gene
• Remove mRNA from β-cells in islets of langerhans
• Incubate mRNA with reverse transcriptase
• Produces complementary single stranded DNA
• This is converted to double stranded DNA – insulin gene
• DNA polymerase and supply of free nucleotides added to single
stranded DNA to produce a copy of the original DNA called cDNA
• Unpaired nucleotides are added to each end of the cDNA to create
sticky ends complementary to cut plasmid
Genetically engineering insulin
• Plasmids cut open with restriction
enzymes are mixed with cDNA
genes
• DNA ligase seals plasmid with new
gene – now a recombinant
• Plasmids mixed with bacteria, some
of which take up new plasmids, but
not all
• Bacteria grown on agar plates to
create colonies of clones
Not all bacteria take up plasmid 
• Some bacteria do not take up plasmid
• Some bacteria take up plasmid that has not
sealed with new cDNA and resealed with out
it
• Some will take up recombinant plasmid –
these are transformed bacteria 
• Can’t tell the different just by looking at them!
Identification of transformed bacteria by
replica plating
• Plasmids are chosen that carry genes that make them resistant to 2
different antibiotics (ampicillin and tetracycline)
• These resistant genes are known as genetic markers
• The bacteria E.Coli are susceptible to both these antibiotic chemicals
• Plasmids are cut by restriction enzymes that has its target site (where it
cuts) in the middle of tetracycline resistance gene
• This means that if the required gene is taken up, the gene for tetracycline
resistance is broken up and fails to work, but the gene for ampicillin
resistance does still work
How replica plating is used
• Bacteria are grown on nutrient agar so all bacteria grow,
forming colonies
• Some cells are transferred onto agar that contains ampicillin,
so only those that have taken up the plasmid with the gene to
kill the bacteria will grow
• Some cells from these colonies are transferred onto agar that
has been made with tetracycline, so only those that have ten
up a plasmid that does not have insulin will grow (the gene
has not been cut)
How replica plating is used
• By keeping track of which colonies are which, it can be
deduced where the insulin gene is.
• Bacteria growing on ampicillin agar, but not on tetracycline
agar must contain the plasmid for the insulin gene
• Colonies can be identified and grown on a larger scale
• Bacteria producing insulin are harvested for later use
Questions
A scientist has genetically engineered some bacterial cells to contain a
desired gene and a gene that gives resistance to penicillin. The cells
were grown on agar place and then transferred to a plate containing
penicillin.
(a) Explain why the scientist thinks colony A contains transformed
bacterial cells, but colony B does not. (2)
(b) Explain how the scientist might have inserted the desired gene
into the plasmid (3)
(c) Explain why being able to take up plasmids is useful to bacteria (2)
Answers
(a) Colony A has grown on the agar plate containing penicillin (1 mark) so it
contains the penicillin-resistance marker gene, which means it contains
the transformed cells (1 mark)
(b) The plasmid vector DNA would have been cut open with the same
restriction endonuclease that was used to isolate the DNA fragment
containing the desired gene (1 mark). The plasmid DNA and the gene
(DNA fragment) would have been mixed together with DNA ligase
(1mark) DNA ligase joins the sugar-phosphate backbone of the two bits
of DNA (1 mark)
(c) It is useful for bacteria to take up plasmids because the plasmids may
contain useful genes (1 mark) that increase their chance of survival (1
mark)
One product manufactured using microorganisms is insulin. The process involves genetically
engineering bacteria to synthesise human insulin.
(i)
Describe how the isolated human insulin gene is inserted into a bacteria plasmid.
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(ii)
Suggest two ways in which the bacteria which take up the modified plasmids can
be identified.
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[2]
[Total 6 marks]
(i) plasmid cut by restriction enzyme;
at specific sequence;
same enzyme as used to cut (insulin) gene;
sticky ends / described;
ref. complementary sticky ends;
ligase seals (sugar-phosphate) backbone / AW;
(ii)
max 4
credit any two from the following:
1 antibiotic resistance (gene) introduced and survivors have plasmid;
2 fluorescent marker (gene) introduced and glowing bacteria have plasmid;
3 identify bacteria producing insulin using antibodies;
2
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