بهینه‌سازی بیان سم شقایق دریایی (فراگاسیاتوکسین C) در باکتری E.coli سویه BL21

Background and purpose: Sea anemones of the genus Actinia are commonly found in the intertidal zone of the northern rocky coast of Spain. Actinia fragacea is one of the most important species that secretes a toxic protein, fragaceatoxin C (FraC), for defense and hunting purposes. The aim of this study was transformation of plasmid containing the gene encoding the FraC to Escherichia coli bacterial strain BL21, and identifying optimum expression condition. Materials and methods: In this experimental study, chemical transformation was conducted by washing the bacteria with cold calcium chloride to take up the plasmid containing the FraC gene. To ensure the transformation of plasmids, bacteria were grown on a medium containing an appropriate antibiotic (ampicilin). For greater certainty, the minipreparation of transformed plasmids was done using GF-1 plasmid extraction Kit. The plasmids were then went under digestion check using NcoІ and Hind ІІІ enzymes and PCR check. After confirming the presence of a plasmid containing FraC in the host bacteria, the bacteria were induced by 1mM IPTG in different times at 20 ºC to find the optimum expression conditions. After collecting bacteria expressing FraC at different times, its expression was visualized on Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Results: Our results showed that the plasmid containing the FraC was successfully transformed. Also SDS-PAGE results indicated that the FraC was successfully expressed at times of 6 and 8 h using 1mM IPTG at 20 ºC. Conclusion: According to the results, both steps of chemical transformation had significantly influenced the transformation efficiency. Following the principles and selecting optimum temperature could help us in finding the optimum conditions for expression of each specific protein in bacteria.