Gateway Cassette

In the Gateway cloning is a product marketed by Invitrogen, compared to conventional cloning strategies very efficient cloning technique. After a short time of up to 99 % positive transformants can be obtained.

The technique is based on the sequence-specific recombination system of the phage λ ( a bacterial virus), the integrated with the help of certain enzymes its DNA into the genome of the bacterium Escherichia coli. The integration process ( lysogeny ) is in this case of two proteins catalyzed: the integrase (Int) of phage lambda and the E. coli IHF protein subunits a and b IHF (Integration Host Factor ). This reversible process requires specific sequence segments ( the attachment sites or recombination sites - German about joining or recombination ) in the target DNA between which can be inserted to transfer genetic information. For their integration joints are also required to be transferred to the ends of these DNA fragments. For excision ( lysis) of the DNA fragment, in addition, the enzyme Exzisionase is required.

Implementation

Cloning basically consists of three steps.

1 ) Generation of attB - PCR product

In order to express the gene of interest is ultimately successful, it must first be provided with the attB recombination sites. These are composed of 25 base pairs, and are recognized by the enzymes involved in recombination. Thus, the gene of interest is placed in the correct reading frame in the expression vector, the connection points for both ends of the DNA segment were differently constructed in such a way that they receive the correct orientation after integration into the donor vector.

The connection points may be added, for example, by PCR with specific primers bearing at the 5'- end of the attB sites in the gene to be expressed.

2 ) Generation of Entry Clones

The second step consists in the replacement of the contained in the donor vector ccdB gene with the PCR product (BP reaction). Wherein the ccdB gene is a suicide gene, whose gene product at the bacterial strains commonly used in laboratories is toxic, by inhibiting gyrase, and carrying at its two ends the attP joints.

Accordingly, should after successful transformation only those bacteria be viable, in which the exchange of the ccdB gene is against the PCR product. In addition, available from Invitrogen donor vectors carry antibiotic resistance. This double selection is one of the reasons for the high effectiveness of this cloning method. At the taking place between PCR product and donor vector BP reaction, the enzymes integrase and IHF a / b are involved. The attB1 -site reacts specifically with this attP1 and attB2 with attP2, whereby the attL sequences are generated in the resulting entry clone. As examples, the product flanked with the attR sites suicide gene is obtained.

3 ) generating the expression vector

The third step consists of the reaction between the Entry clone, and the target vector ( LR reaction). If a gene fusion to be brought about, the target vector 5 'or 3' of the attR sites can wear the appropriate sequence. Depending on whether the gene product of an N-terminal or C- terminal fusion to be brought about. For example, the target vector carrying the sequences for fluorescent proteins, if interaction is to be tested by FRET on a protein-protein. In addition, the target vector containing the flanked by attR sites ccdB gene. By 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, is needed for the additional enzyme Exzisionase ( Xis ). To re-enable a double selection, wearing the Target Vector another antibiotic resistance as the Entry Clone. As the case of a product - the ccdB suicide gene results from the LR reaction except the expression vector plasmid carrying. These transformants perish and only the desired transformants come to growth.