產(chǎn)品信息

     The Expresso Rhamnose Cloning and Protein Expression Systems are designed for fast, easy, and efficient directional cloning and expression of PCR-amplified genes using Expressioneering Technology. Expressioneering Technology uses in vivo homologous recombination to seamlessly clone PCR amplified DNA into specially designed expression vectors without the need for enzymes or purification steps. A single host strain is used for both stable cloning and controlled protein expression, making Expresso Rhamnose the fastest cloning and expression systems available. The systems come complete with pre-processed expression plasmids and competent cells, supplied in single transformation vials.
優(yōu)點(diǎn)：
 ? Five-second, directional, enzyme-free PCR cloning! 90% recombinants!
 ? Single competent cell host strain for both cloning and expression.
 ?Tighter expression control with tunable Rhamnose (rhaPBAD) promoter.
數據

Figure 1. Expressioneering Technology uses in vivo homologous recombination to seamlessly clone PCR amplified DNA into specially designed expression vectors without the need for enzymes or purification steps. The desired insert is simply amplified with primers that include 18 bases that overlap with the ends of the Expresso? vector. The unpurified PCR amplicon is then mixed with the pRham expression plasmid and the high-efficiency competent cells provided, and directly plated on appropriate media.

Figure 2. pRham expression vectors. RBS, ribosome binding site; ATG, translation start site; Stop, translation end site; Kan, kanamycin resistance gene; ROP, Repressor of Priming (for low copy number); Ori, origin of replication. CloneSmart? transcription terminators (T) prevent transcription into or out of the insert, and a terminator follows the cloning site. The 6xHis affinity tag is fused to the amino terminus (pRham N-His) or at the carboxyl terminus (pRham C-His) of the expressed target protein. Also available: pRham N-His SUMO Vector for enhanced soluble protein expression with cleavable SUMO solubility tag.

Figure 3. Tuning recombinant protein expression levels with rhamnose induction. The pRham C-His Kan Vector containing a gene encoding a blue fluorescent protein (BFP) was transformed into E. cloni 10G cells. An uninduced starter culture was inoculated to a starting OD600 of 0.8 into culture tubes containing LB media with 30 μg/ml kanamycin and the indicated concentrations of rhamnose (0 to 0.2% w/v) or 2% glucose. After overnight incubation at 37°C, samples were harvested by centrifugation, lysed in SDS-PAGE loading buffer, and analyzed by SDS-PAGE. The Coomassie-blue stained gel shows total cellular protein. Protein expression levels are responsive to rhamnose concentrations between 0.001% and 0.2%.
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