Principle

The CYS21 bacterium, included in the StabyCloning kit, 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.

Results

Using the StabyCloning^TM kit, 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 CYS21 bacteria and the antidote properties.

Kit Content

The kit contains linearized plasmid DNA of the pSTC1 vector, competent bacteria (chemically or electro), ligase, regeneration medium, sequencing primers, positive control, and the instruction manual. Additional competent bacteria can be purchased separately.

Benefits

• 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.