Gateway cloning
In the Gateway cloning is a method for cloning . It has a higher cloning efficiency compared to cloning by restriction and ligation . After a short time, up to 99% positive transformants can be obtained.
The technology is based on the sequence-specific recombination system of phage λ (a bacterial virus), which uses certain enzymes to integrate its DNA into the genome of the Escherichia coli bacterium . The integration process ( lysogeny ) is in this case of two proteins catalyzes: the integrase (Int) of lambda phage and the E. coli - protein subunits IHF a and b IHF ( integration host Factors ). This reversible process requires specific sequence sections (the attachment sites or recombination sites ) in the target DNA, between which the genetic information to be transmitted can be inserted. For their integration, connection points are also required at the ends of these DNA segments to be transferred. For excision (lysis) of the DNA fragment which is additionally enzyme Exzisionase needed.
execution
Cloning essentially consists of three steps.
1.) Generation of an attB -PCR product
In order to ultimately be able to successfully express the gene of interest , it must first be provided with the attB recombination sites. These consist of 25 base pairs and are recognized by the enzymes involved in the recombination. So that the gene of interest is arranged in the correct reading frame in the expression vector, the connection points for both ends of the DNA segment were constructed differently, specifically in such a way that they receive the correct orientation after integration into the donor vector.
The connection sites can be added to the gene to be expressed , for example by a PCR with specific primers which have the attB sites at the 5 'end .
2.) Creation of an entry clone
The second step consists in exchanging the ccdB gene contained in the donor vector for the PCR product (BP reaction). The ccdB gene is a suicide gene, the gene product of which has a toxic effect on the bacterial strains normally used in the laboratory by inhibiting gyrase , and which has the attP connection points at both ends .
Accordingly, after successful transformation, only those bacteria should be able to survive in which the ccdB gene has been exchanged for the PCR product. In addition, the donor vectors available from Invitrogen are antibiotic-resistant. This double selection is one reason for the high effectiveness of this cloning method. The enzymes integrase and IHF a / b are involved in the BP reaction between the PCR product and the donor vector. The att B1 - site reacts specifically with this attP1 and att B2 with attP2 , thus resulting in the entry clone the attL sequences are generated. The suicide gene flanked by the attR sites is obtained as a by-product .
3.) Generation of the expression vector
The third step consists of the reaction between the entry clone and the target vector (LR reaction). If a gene fusion is to be brought about, the target vector 5 'or 3' of the attR sides can carry the corresponding sequence. Depending on whether an N-terminal or C-terminal fusion is to be brought about in the gene product. For example, the target vector can carry the sequences for fluorescent proteins if FRET is to be used to test for a protein-protein interaction. In addition, the target vector contains the ccdB gene flanked by the attR sites . As a result of the LR reaction between attL1 and attR1 and between attL2 and attR2 , the attB pages are generated again in the expression vector . This reaction corresponds to a reverse BP reaction, for which the enzyme excisionase (Xis) is also required. In order to enable a double selection again, the target vector has a different antibiotic resistance than the entry clone . In addition to the expression vector, the by-product of the LR reaction is a plasmid carrying the suicide gene ccdB . These transformants perish and only the desired transformants grow.