| Products/Services Used | Details | Operation |
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| Gene Synthesis | A synthesized codon-optimized version of the PfAgo gene was ordered from GenScript (Nanjing, China), which was designed as the pET28a-derivative plasmid pEX-PfAgo with an N-terminal His-tag. The expression plasmid was transformed into E. coli BL21(DE3) cells. A 5 mL seed culture was grown at 37 °C in LB medium with 50 μg/mL kanamycin, which was transferred to 1 L of LB in a shake flask containing 50 μg/mL kanamycin. The cultures were incubated at 37 °C until OD600 values of 0.8-1.0 was reached, and protein expression was then induced by addition of isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, followed by incubation for 16 h at 20 °C. Cells were harvested for centrifugation for 20 min at 6,000 rpm, and the cell pellet was collected for later purification. | Get A Quote |
Thermophilic Argonaute proteins (Agos) can function as endonucleases via specific guide-target base-pairing cleavage for host defense. The ability to cleave target DNA sequences at any arbitrary sites endows them with reprogramed DNA capacity. Here, we identify that an Ago from the hyperthermophilic archaeon Pyrococcus furiosus(PfAgo) shows a stepwise endonuclease activity, which is demonstrated by the double strand DNA cleavage directed by a single guide DNA rather than canonical one pair of guide DNAs. We reveal that the cleavage products with 5’-phosphorylated ends can used as the renewed guide which is capable to induce next round of cleavage to complementary sequences of target DNA. By combining the Pf... More
