Transcript Cloning and selection

Transformation
•Movement of DNA into
bacteria
•Can use plasmid as vector
•Some bacteria are naturally
competent and take up DNA
from their surrounds
•Most bacteria are not,
including E coli and we have to
make it competent and get it
to take up DNA from its
environment
•Competency is a physiological
state where bacteria can take
up DNA from their environment
•Abuse bacteria with cold CaCl
When do the cutting and sticking of plasmid and foreign
DNA there are several possible outcomes
1. Successful sticking of the plasmid and foreign DNA
2. Recircularization of plasmid without the foreign DNA
3. Circulization of plasmid with other plasmids or several
inserts to make huge circular molecule
4. Many inserts sticking together to make long linear
molecule
All of these outcomes occur and could be taken up into
bacteria
Possibility 3 is less likely to happen as big plasmids are
more difficult to take up and also less stable when they
are taken up
Possibility 4 can occur but linear DNA is usually broken
down very quickly in a bacterial cell as it is recognised
as non self
Selection
A mechanism is required to select for the bacterial cells that have taken
up the plasmid with one inserted foreign DNA piece
Usually bacterial cells are used that are sensitive to a particular
antibiotic
The recombinant plasmid that is used usually carries antibiotic
resistance and also a gene for Beta galactosidase.
Beta galactosidase is an enzyme that acts on a substrate called X gal
to yield a coloured product.
The foreign DNA is inserted into a site in the middle of the beta
galactosidase gene. This disrupts it and makes it inactive and
unable to perform the colour reaction with X gal.
Nutrient agar with ampicillin
and X gal
Plasmid vectors
•small circular dsDNA molecules capable of autonomous replication inside cells.
•Usually of bacterial origin (there are some eukaryotic plasmids).
Designed Plasmids must have:
1.origin of replication (Ori),
2.Termination seqs
3.Selective marker (usually antibiotic
resistance gene) to select for bacteria
containing plasmid.
4.Polycloning site (unique restriction
target sites useful for inserting donor
fragments
5.Many plasmids allow for blue/white
selection for distinguishing between
recombinant vs nonrecombinant
plasmids
6.polycloning site inserted inframe
into beta-galactosidase gene
(product of this gene converts
colorless X-gal into blue product)
Fragments inserted into this
polycloning site disrupt function of bgalactosidase gene product, resulting
in white colonies.
• plasmids used as vector when fragments
of DNA to be inserted are smaller than 20
kb.
• Some plasmid vectors constructed such
that cloned genes can be transcribed and
translated. These are called expression
vectors .
Types of Naturally occurring Plasmids
– All plasmids carry genes for their own replication – what other genes?
– No apparent function – cryptic plasmids
– for conjugation
– For antibiotic production
– Bacteriocin production
– Resistance genes
– Virulence genes
– Physiological functions: degradation of chemicals/herbicides, production
of acetone, nodulation in symb N2 fixation
Resistance Plasmids
• R plasmids – resistance to antibiotics and other growth inhibitors
• Genes encode proteins that inactivate Ab or affect its uptake
• Can transfer resistance via conjugation
• Many drug resistance elements are on transposons
• Plasmid R100 carries resistance to sulfonamides, streptomycin,
spectinomycin, chloramphenicol etc and can transfer itself to
Klebsiella, Proteus, Salmonella and Shigella
Transfer of DNA between Cells
– In prokaryotes DNA transferred to recipient cell by 1
of 3 processes:
– Transformation: involves donor DNA free in
environment
– Transduction: donor DNA transfer mediated by a virus
– Conjugation: involves cell to cell contact between
donor and recipient i.e. acquisition directly from
another bacterium