In vivo gene cloning 19 June 2024

Use of vectors
Learning objectives
Explain the importance of sticky ends
Explain the insertion of DNA fragment into a vector
Explain the introduction of the vector into host cells

Starter
1. Recall the three main ways that DNA
fragments can be isolated
2. Review the process of using the ‘gene
machine’
The gene machine
1. What information must be fed into the computer before
the gene machine can begin? (1 mark)

2. What two aspects is the sequence checked for before

the process can begin? (1 mark)

3. What are oligonucleotides? (1 mark)

4. By what process is the final gene replicated? (1 mark)

5. Give two advantages of using the gene machine (2 marks)

DNA Technology

1. Isolation – of the DNA containing the required

gene
2. Insertion – of the DNA into a vector  We will now look at this stage
3. Transformation – Transfer of DNA into a
suitable host
4. Identification – finding those host organisms
containing the vector and DNA (by use of gene
markers)
5. Growth/Cloning – of the successful host cells
Once the fragments of DNA with the desired gene
have been obtained, they must be cloned either by
1. In vivo cloning - transferring the fragments to
 We will be
a host cell using a vector
focusing today on
2. In vitro using the ‘Polymerase Chain Reaction’ in vivo cloning
Sticky ends are important in the insertion of
What
the type
DNA as of end dofor
it allows youcomplementary
think is useful base
for the purposes of inserting the DNA
pairing:
fragment into a vector? Why? Once complementary bases of
- Recognition sites staggered
sticky ends are paired up, DNA
- Cut ends are single stranded and a few ligase is used to bind the
nucleotide bases long – They are sugar-phosphate of the two
complementary to those at the other side DNA sections together
- All fragments cut by the same
endonuclease are therefore complementary
Preparation of DNA
 Addition of extra lengths of DNA for
RNA polymerase to function (to attach to
the DNA for transcription)
 This binding site for RNA polymerase is
called a promoter – this attaches the RNA
polymerase and so begins transcription
 Another region of DNA is necessary to
release the RNA polymerase (to end
transcription) = terminator

Animation Quiz 1 - Cloning a Gene

https://www.kerboodle.com/api/courses/15056/interactives/111859.html
Insertion into a vector So far…
 Fragment of DNA isolated
 Promoter and terminator regions
added

• Now, DNA fragment must be joined to the vector – used to

transport the DNA into the host cell
• Most common vector = plasmid (circular lengths of DNA in
bacteria separate from main bacterial DNA) tance
ti c re si s
s f or a n tibio
s c on ta in gene
Plasmid
What do you think are used to break/cut
Restriction the plasmid loop at these
endonucleases!
antibiotic-resistance genes?
The same one that cuts out the DNA fragment – sticky ends are complementary

DNA fragments and opened plasmids are mixed and

become incorporated – this join is made permanent by
DNA ligase
Summary
Transformation This is the introduction of DNA into host cells

 The bacteria, plasmids and calcium are mixed together.

 Calcium ions, plus changes in temperature, make the bacterial
membrane permeable, allowing plasmids to enter the cytoplasm

NOT ALL bacterial cells possess the DNA fragments

because:
1. Only a few bacterial cells (less than 1%) take up the
plasmids
2. Some plasmids would have closed up without
incorporating the DNA fragment
3. The DNA fragment ends may have joined together to
form its own plasmid
Identification of successful transfer
This uses the idea of antibiotic resistance in bacteria

Using p537, answer the following questions:

1. How do bacteria resist the effects of antibiotics? (1 mark)
2. Where are the genes for antibiotic resistance found? (1 mark)
3. For which two antibiotics does the R-plasmid carry resistant
genes? (2 marks)
4. Outline how we can use antibiotic resistance of bacteria in order
to identify which bacterial cells have taken up the plasmids (4
marks)
5. Why is the above process in itself not sufficient to identify
successful transfer of the recombinant DNA? (1 mark) And what
do we use to overcome this issue? (1 mark)
Marker genes

3 main types of marker genes:

1. Antibiotic-resistance marker genes (use of replica plating)
2. Fluorescent markers
3. Enzyme markers

HOMEWORK – Research and summarise the three main types of

marker genes ready to answer questions on this next lesson
The Plasmid

The ampicillin resistance gene

is disrupted when the
restriction enzymes cuts open
the plasmid.
Antibiotic-resistance Markers
► The second antibiotic-resistance gene (e.g. resistance
to tetracylcine) is used to identify those plasmids
with a DNA fragment in them.
► If the DNA fragment has been inserted into the
tetracycline resistance gene it will no longer grow on
medium containing tetracycline .
► In order to identify these bacteria we use a process
called replica plating.
Replica Plating

Ampicillin sensitive bacteria – these

have the DNA fragment

►The bacteria on the yellow plate have the plasmid.

►The bacteria which do NOT grow on the green plate
(containing tetracycline) contain a plasmid with a DNA
fragment.
Fluorescent markers
► The gene from jellyfish which
produces Green Fluorescent Protein
(GFP) has been incorporated into a
plasmid.
► If the DNA fragment has been
inserted into the GFP gene, the
bacterial will not glow and can be
identified.
► If the DNA fragment has not been
inserted into the GFP gene, the
bacteria will glow and would not be
used.
Enzyme Markers
►The enzyme lactase turns a colourless
substance (B-galactosidase) a blue colour

►If the gene has been disrupted by the

incorporation of the gene fragment the
substrate will remain colourless.