Our Technology

Delphi Genetics Technology

Modern DNA engineering requires efficient and robust molecular tools. The Staby® Operating System, developed by Delphi Genetics is an original set of bacterial strains and vectors integrating unique patented technologies. The use of these products and technologies allows researchers to reach their goal faster and more efficiently. These technologies can be used in a project of any scale (from laboratory scale to industrial processes).

The Staby® Operating system is very flexible and its elements can be integrated into your favorite tools, allowing you to add the power of the Delphi-Genetics technologies into your own platform.

Technological Context and Concerns

For manufacturing recombinant proteins or DNA, plasmid instability is a significant concern. Typical biomanufacturing processes in prokaryotes require the use of bacterial plasmids as vectors carrying the gene of interest to be over-expressed. It has been demonstrated that the growth of plasmid bearing cells is significantly reduced relative to plasmid free hosts, simply because protein production represents a significant burden on cellular metabolism. This is of significant concern in the Biopharmaceutical Industry as both the yield and the production reproducibility of recombinant molecules are significantly lowered by that fact.

Antibiotic resistance genes are the most common selectable markers used in fermentation processes to avoid plasmid free cells to overgrow the culture. However antibiotics are expensive compounds and they (or their degradation products) can contaminate the biomass or production product. These contaminations are unacceptable from industrial, medical and regulatory perspectives. Consequently, when using antibiotics it has to be demonstrated that the final product is “antibiotic-free”. The assessment of the residual antibiotic levels and if necessary their removal are also costly procedures. Given these facts, the current trend in the Biomanufacturing industry is to forgo antibiotics in the production process altogether. This implies that the production process per se is only loosely controlled, leading to diminishing returns at scale-up (due to plasmid instability) and difficulties to consistently reproduce production levels of a given Biomolecule.

Delphi Genetics' Solution

Several alternative strategies have been developed to minimize the risk that plasmid-free cells overtake the culture. One of the most promising of those strategies relies on the use of postsegretional killing genes (so called poison / antidote genes or selection modules) that induce host killing upon plasmid loss.

Delphi Genetics has designed a novel and highly effective stabilization system, called Staby®, based on the use of selection modules naturally found in plasmids, bacterial chromosomes and bacteriophages. A selection module (like the ccd system used by Delphi Genetics) is typically organized as an operon and composed of two genes: the ccdB selection gene codes for a small stable protein which is toxic for E. coli, whereas the ccdA antidote gene codes for a small unstable protein that neutralizes the toxic protein both transcriptionaly and via protein-protein interactions.

The Staby® system is based on the ccd module.

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The antidote gene has been separated from the selection gene, locating the former on the expression plasmid and the later in the chromosome of the expression strain. Expression of the selection gene is under the control of a promoter which is strongly repressed in the presence of the plasmid. When the plasmid is lost, the antidote protein is degraded and the production of the poison is induced causing cell death

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This unique system allows for the perfect stabilization of the plasmid without the use of antibiotics. Furthermore it ensures that during protein expression every bacterium is carrying the expression plasmid thus enhancing the overall yield of any given protein expression process. If some bacteria lose the vector, they will not obtain a selective (growth speed) advantage but will die. In practice, this additional stabilization technology solves the problem of plasmid instability and insures that upon induction 100% of the bacteria will produce the recombinant protein leading to higher yields of the target protein and less background caused by unwanted proteins (proteins from bacteria lacking the plasmid). Thus, the production of the protein of interest is higher and purer.

Highest Levels of protein Expression

In order to maximize protein expression, the Staby® system was combined to the proven T7 polymerase technology for the over-expression of recombinant proteins: the gene of interest is under the control of the T7 promoter. This combination is called StabyExpress®. The pStaby1 plasmid contains the gene encoding the antidote protein (ccdA) which counteracts the action of the ccdB protein. The SE1 strain, in which the ccdB selection gene is inserted in the chromosome in order to stabilize the pStaby1 plasmid has been engineered by Delphi Genetics as a dedicated bacterial strain for protein expression and is derived from BL21(DE3). Consequently, IPTG has to be used to induce protein expression, an alternative strategy being the use of an auto-inducible medium (such as Delphi Genetics' Staby®Switch medium). Staby®Express ensures that only plasmid-bearing clones can grow whereas every clone losing the plasmid will inevitably die. This unique feature guarantees the highest possible yields from a given amount of bacteria. As the stabilization is purely based on genetic elements (e.g. no special metabolites are required for selection), Staby®Express can be used in any culture medium even synthetically defined media.

Unique Versatility

Because the stabilization relies on the presence of only two genetic elements, one on the plasmid the other in the expression strain, the stabilization technology is readily adaptable into any existing E.Coli based expression platform. Delphi Genetics can customize any vectors and strains by adding the stabilization cassette into the vector and the selection gene into the chromosome of the chosen expression strain. Furthermore Delphi Genetics has developed a comprehensive licensing policy for those who would wish to integrate the stabilization system into an existing platform.

It works for cloning too

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. From the early development of molecular cloning, identifying vectors with an insert has always been a frustrating and time-consuming step for the investigator.

Traditionally clone selection was based on the disruption of a genetic marker (e.g. blue and white screening based on the disruption of the LacZ gene), plasmid retention was based on antibiotic resistance genes. The development of vectors employing selection modules circumvents these issues and permits 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. The ccdB technology 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. Most of the early commercial vector versions 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.

Delphi Genetics has introduced a new and improved positive selection sytem, called Staby®Cloning which uses both the ccdA and the ccdB proteins for the selection process. 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 poison. 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 Ampiciline) 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.

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