Restriction enzyme cloning into an Entry Vector, such as pENTR1A. You have a choice of three molecular approaches to create your Entry Clones.ġ. You can create your own Destination Vectors by adding a complete Gateway Cassette, which includes att sites and the selectable markers, into an expression vector of your choice. The ccdB gene and associated chloramphenicol gene are a universal feature of all Gateway vectors. The same ccdB gene provides negative selection against intact destination vectors. In this case, the plasmid is positively selected for with Ampicillin. The same pattern of positive and negative selection is used after performing LR clonase reactions to create your Expression clone. A ccdB gene, which is a suicide gene and will kill any bacteria that hosts itįollowing the BP reaction, which introduces your fragment of interest between the attP sites, all plasmids transformed will be able to survive Kanamycin selection, but only recombinants, which have lost the ccdB gene will be able to grow.A Kanamycin resistance gene which will positively select for the presence of the plasmid.Gateway cloning takes advantage of both positive and negative selection during the process of propagating vectors and selecting for recombinant plasmids.įor instance, the Donor Vector pDONR201 contains multiple genes for selection including: your fragment of interest is now located between attL sites, and ready for subsequent Gateway Reactions. The product you generate with your Entry Vector or Donor vector is an Entry Clone i.e. In contrast, Donor vectors require you to use PCR to add attB sites to your fragment of interest then use the Gateway BP Clonase reaction. An entry clone is a plasmid carrying a fragment of interest located between attL sites.Įntry vectors depend on conventional restriction enzyme cloning to introduce your fragment of interest between the attL sites. Creation of Expression Clones - LR ReactionĮntry Vectors and Donor Vectors are used to capture a gene or gene fragment of interest, to create an Entry Clone.Designing a Gateway Cloning Experiment Gateway cloning proceeds in two steps: These two recombination events are perpetually reversible. Since Gateway Cloning is moving elements from one plasmid to another, they do not refer to integration and excision, but rather the BP reaction (BP→ LR) and the LR reaction (LR→ BP). The Gateway system uses both the integration and excision reactions to create an alternative to restriction enzyme cloning. The phage then begins its lytic growth cycle. Upon excision, attL and attR are converted back to attP and attB, and the phage DNA is removed from the bacterial chromosome. If the phage senses that the bacteria is under stress, it will excise itself. After insertion, the recombination sequences are now called attL (left) and attR (right). Recombination between the B and P elements results in new recombination sequences flanking the inserted phage DNA. In this diagram, you can see that the integration system involves the specific DNA sequences found on the phage, attP-sites, as well as sites found on the bacterial genomic target, attB. Diagram of phage lambda behavior as part of a gateway cloning process.
0 Comments
Leave a Reply. |