Sharpened tools for life-science discoveries
The StabyExpress T7 kit contains all the necessary elements for cloning of a gene-of-interest and its expression in Escherichia coli. The kit combines two technologies (T7 expression and Staby plasmid stabilization) that allow high-yield protein expression and standardization of the production-protocol. Vectors with His-tag and/or GST-tag are available. Additional CYS21 and SE1 competent bacteria can be purchased separately.
Plasmid instability is a significant concern in protein production. Typically, protein-production processes require the use of bacterial plasmids as vectors carrying the gene to be over-expressed. It has been demonstrated that the growth rate of plasmid-bearing cells is significantly reduced relative to that of a plasmid-free host, simply because protein production (corresponding to the gene-of-interest over-expression) represents a significant burden on cellular metabolism. Antibiotic-resistance genes are the most common selectable markers used in fermentation procedures to avoid plasmid-free cells to survive and dominate the culture. However, contamination of the product or biomass by antibiotics (or genes encoding an antibiotic resistance) is unacceptable from a medical or regulatory perspective (cf. FDA recommendations). Our new stabilization system is based on the use of antidote/poison genes naturally found in plasmids, chromosomes, and bacteriophages. The ccdB selection gene codes for a small stable protein (about 100 amino acids) whereas the ccdA antidote gene codes for a small unstable protein (about 90 aa) that neutralizes the selection protein. These genes are extensively used in DNA cloning technology and are only active in enterobacteriacae (E. coli, Salmonella).
In our plasmid-stability system, the antidote gene is introduced in the plasmid DNA under the control of a constitutive promoter. On the other hand, the selection gene is introduced in the chromosome of the bacteria. Expression of this selection gene is under the control of a promoter strongly repressed by the antidote protein. Hence, when the plasmid is present in the bacteria, the poison is not produced. On the other hand, when the plasmid is lost, the antidote is degraded and the production of the toxin is induced, causing cell death.
The StabyExpress system is compatible with the use of an auto-inducible medium asStabyTMSwitch.
The system was tested in E.Coli and the results show a perfect plasmid stabilization of several vectors unstable without our technology. In the StabyExpress T7 kit, this technology is applied to protein production using the T7 promoter. The figure compares the production of a 69kDA protein with and without the StabyExpress technology.
Higher plasmid stability = more proteins
Using StabyExpressTM, the plasmid is perfectly stable before and after the induction period. On the contrary, using the conventional strain BL21(DE3), the plasmid is not stably maintained . Consequently the production of the protein of interest is higher (3 to 5 times) with StabyExpress (lanes c,g and h) than using a conventional system with (lane f) or without (lane d) antibiotics. lanes a, b and e are uninduced conrols. Note that no over-production of the antidote is detectable.
The StabyExpressTM T7 expression kit allows (i) cloning of your gene of interest into a stabilized plasmid and (ii) T7 expression of your protein of interest. Each kit contains plasmid DNA of the pStaby vector (either pStaby1.2 or pStabyGST1.2), CYS21 competent bacteria for cloning, SE1 competent bacteria for expression, regeneration medium, sequencing primers, expression control, and the instruction manual. Additional CYS21 or SE1 competent bacteria can be purchased separately.
Benefits of the StabyExpress T7 kit: