Principle

Some inserts, when cloned into a classical vector, may cause instability of this vector resulting in low yield of DNA or protein production (for protein expression vectors). The reasons for this instability are, for example, the toxicity of the insert and/or the secondary structure and/or the size of the vector. The GetStaby^TM kit provides, on one hand, a plasmid containing an antidote gene (ccdA) under the control of a constitutive promoter and, on the other hand, an E. coli strain (CYS21) that contains, in its chromosome, the selection gene (ccdB) under the control of a promoter strongly repressed in the presence of the plasmid. Contrary to classical strains, if some CYS21 bacteria lose the vector, they will not obtain a selective (growth speed) advantage, but will die. In practice, this means that at any time during bacterial growth, 100% of the CYS21 bacteria will carry the vector even in the absence of antibiotics. These bacteria will yield high amounts of DNA or protein. Adding the Staby^TM cassette (the DNA fragment encoding the CcdA antidote protein) to a vector results in stabilization of this vector in CYS21 (or any other strain containing the ccdB-selection gene in its chromosome).

The Staby^TM cassette is isolated from the pGetStabyA vector using restriction sites or PCR amplification.

The insertion of the Staby^TM cassette into any vector is easily selected using the antidote properties of this cassette and the CYS21 bacteria (encoding the CcdB selection protein). Then, the stabilization cassette linked to any part of the vector can be easily exported to other vectors and selected using the same properties.

Kit Content

The GetStaby^TM kit allows you to stabilize your own vectors. It is compatible with any expression system. The kit contains the plasmid DNA of the pGetStabyA vector, the competent (chemically- or electro-) CYS21 bacteria for cloning and selection, the regeneration medium, the oligonucleotide primers for sequencing or PCR amplification, and the instruction manual.

Benefits of the GetStaby kit

• The DNA or protein production is increased using the stabilized vector (3 to 5 times);
• Plasmids are perfectly stabilized without the use of antibiotics;
• The insertion of the stabilization cassette into any vector is easily selected using the antidote properties;
• When performing protein expression, the background of “parasite proteins” is reduced;
• The GetStaby^TM system is compatible with any vector;
• The GetStaby^TM system is usable in any culture medium (LB, SOC, SOB, YT or minimal media supplemented ith carbon source such as glucose or glycerol,...);
• When using AmpR plasmids, no “satellite” colonies are observed even after extended incubation;
• The stabilization cassette (linked to any part of the vector) is easily re-exported to other vectors.