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MBBT deel 2, MLS studie
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HC7: Vectors for gene cloning an strategies to identify genes in E.coliWhy cloning?
Cloning falls under the protein to gene relation within the Gene-
protein-function pyramid.
Cloning can be used to amplify different proteins, DNA or other
molecules.
Basic principles of cloning
Cloning strategy: design of the correct constructs
First you’ll have to design the correct construct, for example,
you’ll need to produce a DNA sequence with the correct gene
and need to incorporate this into the right vector.
Cloning strategy: restriction enzymes
With the use of restriction enzymes you are able to cut
DNA at the desired place to add a gene. You’ll want to
avoid intermolecular association, this is when the
vector binds to itself, without the insert, with
intramolecular association, the vector and the insert
will bind to each other. To prevent the forming of
plasmids without insert you can make use of alkalic
phosphatase. A vector with phosphorylated ends are
able to bind with the aid of ligase, by using the alkalic
phosphatase the 2Pi groups are removed and the vector
isn’t able to be sealed shut anymore.
When you add your phosphorylated insert, ligase will
be able to seal the vector shut again.
Cloning strategies: linker molecule
Sometimes it is not possible to use restriction eznymes
to insert a gene, so you’ll need to make use of linker
molecules, these are small pieces of DNA where a
restricition site is present. You can also make use of adaptor molecules, these cannot contain
phosphorous groups because they’ll self link. Make use of T4 ATP to remove/add phosphorous
groups.
Cloning strategies: DNA extension using terminal deoxytransferase
Cut a piece of DNA from the insert so you’ll have a small DNA overhang (exonuclease). Add a poly
A-tail making use of dATP on one strand and a poly T-tail using dTTP on another strand. This makes
them complementary and allows ligase to seal the strands together.
,Cloning strategies: PCR amplification and
digestion with restriction enzyme
To perform a PCR you’ll need to make use of a
primer, if you add a restriction site to this primer
you will be able to insert it into a vector.
Downside is that the amplified DNA could not
be homologous
Cloning strategies: recombination
Circular DNA is able to recombine with
genomic DNA of a bacteria, by making
use of recombinase. You need to have
specific sequences, attP & attB
Cloning strategy: gateway cloning
You’ll need a destination vector, has a
specific function, it contains information
to make use this vector to create a
protein. and an entry clone, this contains
the cDNA where you’re interested in
Transformation
Getting DNA integrated into another cell can be achieved by CaCl2 treatment, which binds the
plasmid DNA to the outer membrane of a bacterium, heating the solution to 42 degrees causes the
plasmid to be inserted.
Using electroporation, using an electric field causes small pores to occur within the bacterial
membrane, which allows foreign DNA to be integrated
Selection of a recombinant
Antibiotics
Antibiotics can be used to test if the newly formed plasmid contains your gene of interest, by adding a
gene that allows for antibiotic resistance and adding an antibiotic to your growth medium, only the
bacteria that contain your gene and the R-gene will survive
Counterselection
Take a plasmid with 2 antibiotic resistance genes, for example ampicillin and tetracycline resistance, if
you add a gene within the tetracycline gene, the bacteria will no longer be resistant to tetracycline,
which will allow you to filter out the right plasmids with the right genes by first growing the them on
an ampicillin plate and then touching another plate with tetracycline, some groups will stop growing,
these are your bacteria with the right gene inserted
, Colouring, blue/white screening
Using the a plasmid with ampicillin resistance and a lacZ gene. lacZ allows a white colouring o be
converted into a blue colouring agent. If you insert DNA within the multiple cloning site, which
contains the LacZ gene.
Blue means a complete LacZ gene
White means an incomplete LacZ gene
Selection of recombinant using toxin/antitoxin
Toxin (CcdB) antitoxin (CcdA) systems are part of a plasmid that forces the cell to keep the plasmid in
the cell . Toxin is stable, antitoxin is instable, if plasmid is lost, antitoxin expression gets too low to
inhibit the toxin, cell dies.
Create a plasmid that has a toxin and antitoxin gene, the bacterium will produce a toxin and antitoxin,
inactivating the toxin. If you insert a gene into the antitoxin gene, the antitoxin will be deactivated and
leave, leaving only toxin to be produced.
Different kind of plasmids:
Plasmid survival/maintenance
Plasmid incompatibility
The rule is that two plasmids with the same origin of replication cannot be
kept together, origins can also be incompatible, this means that during cell
division, the plasmids do not replicate and both end up in a different
daughter cell. Often 1 plasmid is more stable or grows faster etc.
F-episome
A plasmid that contains an F-episome produces both a toxin and an
antitoxin. If a plasmid is lost, after replication. The cell without the plasmid
only has toxin lift, which kills the cell. This is called post segregation
killing
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