Learning Outcomes - Week 5 - Cloning genes Flashcards
Learning objectives: what components do cloning vectors contain? Describe them and their functions
- Plasmids contain origin of replication (ori):
- Sequence recognised by replication proteins including DNA polymerase
– Allows replication in E. coli cell
– Determines copy number of plasmid in host bacterial cell (3000 copies/cell) - selectable marker gene
– Why? To clone DNA target, our recombinant plasmids (carrying foreign DNA)
will be added to a tube containing several billion bacteria
- But only some host bacteria will be transformed
- Need to separate transformed cells from non-transformed cells
– Antibiotic resistance gene included
- E.g., ampicillin, many alternates exist
- Tube of transformed and non-transformed bacteria layered on agar
plates containing antibiotics
- Only cells transformed with plasmid will grow - cloning site 1:
– What?
* Allows foreign DNA to be inserted into plasmid
* A sequence that contains multiple restriction enzyme sites
– AKA: multiple cloning site or MCS; polylinker
* Only location of that RE site in plasmid
– How?
* Cut and paste mechanism employed – compatible ends
* Discuss below - cloning site 2:
– What does an MCS look like?
* Sequence of unique cut sites (6 bp) for restriction enzymes
– Means those sites occur nowhere else in plasmid
– Each plasmid vector has different MCS
What are restriction enzymes? Example?
(See image for what they are)
Example is EcoRI
Restriction enzymes: How they work in general terms (specificity) Give example, what makes it and to do what?
How do they work?
Restriction enzymes:
– EcoRI is an example.
– Host bacteria:
* Make restriction enzymes to target foreign invading DNA: viruses and
other bacteria
* Protect themselves by modifying own sites with methyl group
– Modification: methylases add methyl group to base in site
See image for how they work
What are the different DNA ends that they can create (3’/5’ overhangs, blunt)
Explain whether created DNA ends are ‘compatible’ in a cloning procedure
What is DNA ligase?
DNA ligase: Function of the enzyme and why it is used in a cloning procedure
Transformation and colony screening:
How does transformation work?
Plasmid inserted, contains an Phillip resistance (therefore any bacteria that don’t take up the plasmid for).
Any bacteria that that are not recombinant (don’t take the gene of interest) the LacZ which is in the MCS, which is a reporter gene will act on beta galactosidase to convert Xcal into a coloured product and cause a blue colour to appear.
If the plasmid contains the gene of interest, it sits within the MCS as well and inactivates the LacZ resulting in the LacZ not giving rise to beta galactosidase which cant then convert Xcal into a computer product therefore no colour is seen!
Transformation and colony screening:
How can we screen for successful transformants? -> insertional inactivation, DNA
sequencing
Blue-white screening is a rapid and efficient technique for the identification of recombinant bacteria. It relies on the activity of β-galactosidase, an enzyme occurring in E. coli, which cleaves lactose into glucose and galactose
See image for info
Cloning strategies:
Explain using restriction enzyme sites in foreign DNA (to create insert) and cloning vector
Cloning strategies:
Explain using the Taq polymerase ‘A’ overhang
Cloning strategies:
Explain Using restriction enzyme sites that you include in PCR primers
e.
(Example of a question that is very detailed and will not be asked)
What is the origin of the HindIII restriction enzyme?
(a) Hinny (cross between horse and donkey), generation III.
(b) Hindbrain of Capra aegagrus hircus (goat).
(c) Haemophilus influenzae strain D (influenza strain)
(d) Hindleg of Ambystoma mexicanum (axolotl)
c
