Download Lezione 25 - 26 mercoledì 11 maggio 2011

Lezione 25 - 26
mercoledì 11 maggio 2011
corso vettori biologici II
Biotec industriali
ore 14:00 -16:00 aula 6A
A one pot, one step, precision cloning
method with high throughput capability.
PLoS One. 2008;3(11):e3647. Epub 2008 Nov 5. Engler C, Kandzia R,
Marillonnet S.
Current cloning technologies based on site-specific recombination are efficient,
simple to use, and flexible, but have the drawback of leaving recombination site
sequences in the final construct, adding an extra 8 to 13 amino acids to the
expressed protein. We have devised a simple and rapid subcloning strategy to
transfer any DNA fragment of interest from an entry clone into an expression
vector, without this shortcoming. The strategy is based on the use of type IIs
restriction enzymes, which cut outside of their recognition sequence. With proper
design of the cleavage sites, two fragments cut by type IIs restriction enzymes
can be ligated into a product lacking the original restriction site. Based on this
property, a cloning strategy called 'Golden Gate' cloning was devised that allows
to obtain in one tube and one step close to one hundred percent correct
recombinant plasmids after just a 5 minute restriction-ligation. This method is
therefore as efficient as currently used recombination-based cloning technologies
but yields recombinant plasmids that do not contain unwanted sequences in the
final construct, thus providing precision for this fundamental process of genetic
manipulation.
Mettere i
siti per
BsaI
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Legend to figure
General strategy for generation of entry clones lacking internal BsaI
sites. For each gene of interest, two primers are designed to introduce
two BsaI flanking sites (pr1, pr2), as well as one pair of primers for
each internal site to eliminate (pr3, pr4). Column-purified PCR
products pr1–3 and pr2–4 are mixed together with entry cloning vector
(pECV) and BsaI enzyme in restriction-ligation buffer. The mix is
digested for 10 minutes and heat inactivated. Ligase is then added and
the mix is ligated for 10 minutes before transformation in E.coli. All
white colonies contain the expected entry clone, with two flanking BsaI
sites but no internal site. Horizontal arrows represent parts of the
primers identical to the target sequence. Small vertical arrows indicate
the location of the introduced mutation.
Schema della
procedura
si clona in un vettore con
lac-z e si selezionano
colonie bianche o bleu
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Inserimento
siti BsaI
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Leggenda alla figura
General strategy for generation of entry clones lacking
internal BsaI sites.
For each gene of interest, two primers are designed to
introduce two BsaI flanking sites (pr1, pr2), as well as one
pair of primers for each internal site to eliminate (pr3, pr4).
Column-purified PCR products pr1–3 and pr2–4 are mixed
together with entry cloning vector (pECV) and BsaI enzyme
in restriction-ligation...
Golden gate method
Generation of families of construct variants using
golden gate shuffling.
Engler C, Marillonnet S.
Methods Mol Biol. 2011;729:167-81.
The protocol, called Golden Gate shuffling, requires performing the following
steps:1 sequences from several homologous genes are aligned, and
recombination sites defined on conserved sequences; 2 modules defined by the
position of these recombination sites are amplified by PCR with primers
designed to equip them with flanking BsaI sites; 3 the amplified fragments are
cloned as intermediate constructs and sequenced; 4 finally, the intermediate
modules are assembled together in a compatible recipient vector in a one-pot
restriction-ligation. Depending on the needs of the user, and because of the
high cloning efficiency, the resulting constructs can either be screened and
analyzed individually, or, if required in larger numbers, directly used in functional
screens to detect improved protein variants.
sistema semplice
This cloning strategy allows nine separate fragments to
be cloned in a defined linear order into an acceptor vector
in one step, with more than 90% of the colonies obtained
containing the desired construct. Interestingly, this cloning
method is not based on new exotic enzymes or genetic
elements, rather it relies on the use of several previously
known elements that, when used in concert, provide
extremely high efficiency. The most important of these
elements consists of the use of type IIs restriction
enzymes combined with restrictionﾐligation, ensuring a
very high cloning efficiency, and that only the correct
desired construct remains at the end of the restrictionﾐ
ligation. This cloning method not only has applications for
cloning of any construct in general, but is also useful for
DNA shuffling.