Lezione 25 - 26 mercoledì 11 maggio 2011

1. Lezione 25 - 26mercoledì 11 maggio 2011 corso vettori biologici II Biotec industriali ore 14:00 -16:00 aula 6A

2. 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.

3. Mettere i siti per BsaI

4. 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.

5. Schema della procedura si clona in un vettore con lac-z e si selezionano colonie bianche o bleu

6. Inserimento siti BsaI

7. 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...

8. 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.

9. 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.