The Staby®Cloning application is designed for the rapid, precise and efficient DNA cloning of PCR products. The complete cloning procedure is performed in one hour (including plating), the background is basically nil (the bacteria containing vectors without insert are killed), the PCR product is oriented, the plasmid is stabilized, and the export of the insert to another vector is easily selected.
The CYS22 bacterium used in this application contains a natural bacterial poison gene (encoding the selection protein CcdB) in its chromosome. This CcdB protein is only toxic in enterobacteriacae (E. coli, Salmonella). A truncated, inactive, antidote gene (ccdA) is present in the plasmid vector (pSTC1). This vector is linearized at the end of the truncated antidote gene. The ends of the vector are blunt. When a sequence of 14 base pairs (encoding the last 4 codons and the stop codon of the antidote gene) is added to the 5’-end of the DNA fragment to be cloned, the fusion of this sequence with the truncated gene restores an active antidote gene encoding an active protein (CcdA) able to counteract the action of the poison.
The 14-bp sequence is incorporated to the DNA fragment using one modified PCR primer. As illustrated below, this process allows the selection of (i) the recombinant plasmids that incorporate the fragment of interest, and (ii) the orientation of the fragment of interest (only one of the two possible orientations will restore an active, non-truncated, ccdA gene).
Using the Staby®Cloning application, the complete cloning procedure is performed in 1 hour including plating. All the colonies plated are independent clones because no growth phase is required between transformation and plating. All growing bacteria contain the cloning vector with an insert in the correct orientation. Restoration of an active ccdA gene allows the selection of the recombinants and also stabilizes the plasmid into the bacterial population: if some bacteria lose the vector, they will die due to the action of the bacterial poison. After cloning of an insert, it is easy to export the stabilization cassette (antidote gene) together with the insert using the restriction sites in the vector. The insertion into another vector can be selected using the CYS22 bacteria and the antidote properties.
- SPEED: Ligation + transformation + plating in one hour;
- PRECISION: Vectors without insert are eliminated (background<1%) and the DNA fragment is correctly oriented in the vector;
- MOBILITY: Export of [stabilization cassette+insert] is easily selected into other vectors;
- Recombinants are all independent clones (direct plating);
- No “satellite” colonies observed even after extended incubation;
- No risk of background when using AmpR plasmids as PCR template;
- Recombinant plasmids are perfectly stabilized without the use of antibiotics;
- The system is usable in any culture medium.