Technologies using recombinant DNA have become of paramount importance in most biology laboratories today and plasmid toxin/antitoxin (T/A) systems have proven to be very useful by facilitating different key technologies of molecular cloning. Several applications and the underlying technologies are highlighted in this review.Principles of the T/A Technology
Most bacterial strains harbor plasmids that are maintained with remarkable stability. A large variety of plasmids encode systems that act when other control mechanisms have failed, i.e., when plasmid-free progeny is generated during replication. The mechanisms that control plasmid maintenance by T/A loci are well known: the antagonistic regulators that neutralize the toxins are metabolically unstable. Rapid depletion of these unstable regulators occurs in newborn, plasmid-free cells. As the same cells have inherited stable toxin molecules from the mother cell, the toxin will no longer be neutralized by the antitoxin, leading to the killing of the plasmid-free cells. This mechanism effectively reduces the proliferation of plasmid-free cells in growing bacterial populations (1).
The most widely studied T/A system so far is the ccd system located on the F plasmid (2). The ccd system is composed of two genes, ccdA and ccdB, encoding small proteins: the CcdA antidote (8.7 kDa) and the CcdB toxin (11.7 kDa). The CcdB protein acts as a poison because it selectively targets the Escherichia coli DNA gyrase, a bacterial topoisomerase II. Early studies of this T/A system were performed at the Université Libre de Bruxelles (ULB). Today new applications are commercialized by Delphi Genetics SA, a spin off company of the ULB founded by the researchers who developed the use of T/A systems as selectable markers.Positive Selection Vectors
One of the major drawbacks in DNA cloning is the scarcity of the insertion event of the DNA insert into the plasmid. Typically, less than 10% of the vectors circularize with an insert. The 90% of “empty” vectors represent a major background in molecular cloning experiments. From the early development of molecular cloning, identifying vectors with an insert has been a frustrating and time-consuming step for the investigator. The development of vectors employing T/A systems circumvents these issues and permits growth of bacteria harboring insert-bearing plasmids only. Typically, the vectors used in these systems express a gene product that is lethal to certain bacterial hosts. The lethal gene is inactivated by insertion of a segment of foreign DNA and therefore, toxicity is relieved. The most efficient technical solution remains the killing of bacteria harboring an insertless vector, or the selection of bacteria harboring the recombinant vector, the so-called positive selection. T/A systems are of paramount technological significance for positive selection vectors. The ccdB technology (3), pioneered by Researchers at ULB and commercially developed by Delphi Genetics, has proven to be a successful approach for constructing positive selection vectors. The flexibility of the ccdB selection was proven by the system of vectors for different cloning applications based on Delphi Genetics’ ccdB technology sold by the licensee of this technology, Invitrogen Inc. CcdB-based positive selection vectors featuring different copy numbers, a broad-host range and/or transfer properties were developed to facilitate cloning for molecular genetics. The large portfolio of positive selection vectors sold by Invitrogen has nowadays become a standard tool in any normal molecular biology laboratory.
Another system based on the ccdB technology is the Gateway system, also commercialized by Invitrogen. In this system, site-specific recombination is used to insert a gene of interest into the vector, and the recombinants are selected by the replacement of the ccdB gene by the gene of interest. This powerful method allows rapid and efficient transfer of all the genes from an organism from one vector to different vectors (i.e., expression vectors) by automatic sub-cloning.
The next generation of positive selection vectors has recently been introduced by Delphi Genetics. These new versatile vectors are based on a novel application of the T/A properties of the ccd proteins; The StabyCloning™ system from Delphi Genetics uses both the ccdA and the ccdB proteins. The bacteria used in this system contain the ccdB gene in their chromosome. A truncated inactive gene in their chromosome. A truncated inactive version of the antitoxin (ccdA) gene is present in the linearized plasmid vector. When a sequence of 14 base pairs 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 antitoxin protein able to counteract the action of the toxin. The 14-bp sequence is incorporated to the DNA fragment using one modified PCR primer. This system allows for the positive selection of recombinant plasmids only and for the selection of the correct orientation of the cloned fragment in the vector (Figure 1). An additional advantage of this procedure is the speed of the whole procedure: 1 hour until plating.