Plasmid stabilisation technology
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[edit] Introduction
In Molecular Biology and Biotechnology, Plasmid stabilisation technologies are an important issue in particular for the production of recombinant proteins in procaryotic hosts. 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 several key technologies of molecular cloning.
[edit] Principles of plasmid stabilisation technology
Most bacterial strains harbour 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 removed by the antitoxin, therefore leading to the killing of the plasmid-free cells. This mechanism effectively inhibits the proliferation of plasmid-free cells in growing bacterial populations (for a recent review see (1)). The best examined T/A module 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). In presence of the antidote, the ccd promoter is strongly repressed inhibiting the transcription of the operon. The CcdB protein acts as a poison because it selectively targets theE.coli DNA gyrase, a bacterial topoisomerase II. Toxin-antitoxin systems have been useful for three practical applications to date: construction of positive selection vectors, plasmid stabilization, and active biological containment. This review will focus on the two former ones. Genetic analysis of the T/A system and more precisely the ccd operon of F was established at the Université Libre de Bruxelles (ULB) in the 90’s. Based on this research, applications were developed. Today new applications are commercialized several companies including Delphi Genetics SA a Spin off company of the ULB founded by the researchers who pioneered the applications of T/A genes.
[edit] DNA cloning: 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. Generally speaking less than 10% of the vectors circularize with an insert, the remnants recircularizing without an insert (so called empty vectors). Those “empty” vectors represent thus a major background in molecular cloning experiments. From the early development of molecular cloning, identifying vectors with an insert has always been a frustrating and time-consuming step for the investigator. This explains the development of vectors allowing growth of bacteria harbouring 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 harbouring an insertless vector, or the selection of bacteria harbouring the recombinant vector, the so-called positive selection. T/A systems are of paramount technological significance for positive selection vectors. The major advantage, comparing to other selection systems that have been developed, is that the poison of the T/A module are natural small killing proteins with activity that can be neutralized by the antidote. The ccdB technology (3), pioneered by Researchers at the Université Libre de Bruxelles, has been in use for more than a decade in constructing positive selection vectors. Most of the early commercial versions of these vectors were focused on the cloning of PCR generated inserts because the cloning of such inserts leads to an appreciable drop in cloning efficiency (and thus increases the background). This is especially true when a thermostable polymerase presenting a proofreading activity that generates blunt-ended fragments is used. CcdB based positive selection vectors featuring different copy numbers, a broad-host range and/or transfer properties were developed to facilitate cloning for bacterial genetics. Serial Analysis of Gene Expression (SAGE) was originally developed by Genzyme for Gene Expression profiling. Positive selection by ccdB is crucial in this method as it allows for the selection of longer concatemers which is important for the analysis of a large number of genes simultaneously. New positive selection vectors have been introduced by delphi genetics. These new versatile vectors are based on a novel application of the T/A properties of the ccd proteins; Whereas the original positive selection vectors rely only upon the killing activity of the lethal ccdB gene in order to select for transformants, the StabyCloningTM system from Delphi Genetics uses both of the ccd proteins. The bacteria used in this system contain the ccdB gene in their chromosome. A truncated inactive version of the antitoxin (ccdA) gene is present in the linearized plasmid vector. The end of the vector is blunt. 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 (Figure 1). 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 (only one of the two possible orientations will restore an active ccdA gene). An additional advantage of this procedure is the speed of the whole procedure, 1 hour until plating. Due to the innovative positive selection technique the background is virtually nil and the use of antibiotics and the associated pitfalls (e.g. satellite colonies when selecting on ampicillin) is avoided. Another important feature of this technology is that all recombinants are independent clones which allows for direct plating and the system is usable in any culture medium.
[edit] Plasmid stabilization systems for the production of proteins
Plasmid instability is a significant concern in the academic and industrial utilization of microorganisms for protein or DNA production. Usually, these processes require the use of a bacterial plasmid construct as a vector carrying a gene to be expressed. In a fermentation process, cells loosing the plasmidic construct exhibit a higher fitness than construct-free cells, and the former rapidly overcome the later in the bacterial population. Antibiotic resistance genes are the most commonly used selectable markers in fermentation procedures to avoid plasmid free cells to survive and dominate the culture. However several important drawbacks, such as high cost and regulatory issues, are associated with the use of antibiotics. Therefore, several alternative strategies have been developed to reduce the risk that plasmid-free cells overtake a culture; so called plasmid stabilisation strategies. One strategy is to use toxin/antitoxin genes that induce host killing upon plasmid loss. In previously developed stabilization systems using toxin/antitoxin genes (Hok-Sok and parDE T/A systems), the sequence encoding the full operon is cloned into the plasmid to be stabilized. In plasmid-cured bacteria, de novo synthesis of the T/A pair ends, and as a result of antitoxin degradation, the poison is free to exert its toxicity, eventually causing cell death. Unfortunately, these antidote/poison systems can only delay, but not prevent, the takeover of a culture by plasmid-free cells. Indeed, after plasmid loss, dilution of the poison by bacterial growth before complete antidote degradation can allow cells to survive and produce a high-fitness line of plasmid-free cells The StabyExpressTM technology developed by researchers at Delphi Genetics is a highly effective stabilization system based on the use of the ccd toxin/antitoxin genes that addresses these pitfalls. The antitoxin gene has been separated from the poison gene, localizing the former in the plasmid and integrating the later in the bacterial chromosome. The antitoxin gene in the plasmid is under the control of a constitutive promoter whereas the expression of the toxin gene in the chromosome is under the control of a promoter strongly repressed by the antitoxin protein. Thus when the antitoxin is present in the bacteria, the toxin is not produced, whereas upon plasmid loss the toxin is induced causing cell death. This separated-component-stabilization (SCS) (4) strategy: (i) allows for perfect stabilization without the use of antibiotics during the production process; (ii) increases three to five times the recombinant protein production levels; and (iii) does not require any specific modification of the DNA or protein production process or culture medium.
[edit] Additional applications of T/A modules
Takara Bio Inc. of Japan has recently introduced a system based upon the E. coli protein MazF. This protein, a sequence-specific endoribonuclease, cleaves single strandRNAs at ACA sequences. In this system, the transcript of interest which should not contain any ACA sequences (i.e. ACA-less), and MazF are co-expressed in E.coli. Therefore MazF cannot cleave the transcript of interest, but cleaves the ones derived from the host proteins or others at ACA sequences. Thus, after the MazF toxin induction, only the transcript of interest is dominantly translated and only the protein of interest is dominantly expressed.
[edit] Conclusion
The potent bacterial toxins of T/A systems have been fine-tuned by natural selection so that mutants resistant to them arise at very low frequency. Recent bioinformatic studies of bacterial genomes revealed the presence of hundreds of these systems in the chromosomes (5). This demonstrated diversity opens the door for applications in other hosts than the laboratory E. coli strains. Each toxin has evolved in parallel with a specific antitoxin enabling cells to carry and express the toxin gene and yet survive under suitable conditions. This mechanism for controlling cell survival offers biotechnologists a molecular toolbox for selecting recombinants and stabilizing plasmids. The major advantages of T/A systems are: (i) their small size (about 100 amino acids for the poison and 90 amino acids for the antidote); (ii) the efficiency of the toxin selected to exert its activity in bacteria (resistant mutants are rare or nonexistent); and (iii) their broad range of use. Some T/A systems are active in several Gram negative bacteria or even in yeast and mammalian cells. The stabilization system described here should thus be adaptable to other bacterial strains and species (by using appropriate toxin and antitoxin genes) and even in yeast or mammalian cells.
[edit] References
(1) Hayes F. Toxins-antitoxins: plasmid maintenance, programmed cell death, and cell cycle arrest. Science. 2003 Sep 12;301(5639):1496-9
(2) Van Melderen L. Molecular interactions of the CcdB poison with its bacterial target, the DNAgyrase Int J Med Microbiol. 2002 Feb;291(6-7):537-44.
(3) Bernard et al. Positive-selection vectors using the F plasmid ccdB killer gene. Gene. 1994 Oct 11; 148(1):71-4.
(4)Szpirer et al. Separate-component-stabilization system for protein and DNA production without the use of antibiotics. Biotechniques. 2005 May; 38(5):775-81.
(5) Pandey et al. Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes. Nucleic Acids Res. 2005 33(3):966-76.2005.
(5) Lipps G (editor). (2008). Plasmids: Current Research and Future Trends. Caister Academic Press. ISBN 978-1-904455-35-6 .
