The Optimization And Application Of Electroporation Conditions And Primary Study Of Expression Vector For Akthrobacter Simplex

Arthrobacter simplex is a microorganism that is used for steroid drug biotransformation of cortisone acetate(CA)to prednisone acetate(PA).Since the unclear genetic information and the lack of available transformation system and expression plasmids,the molecular modification of A.simplex is largely limited.In this study,we made a scientific optimization of electrotransformation for A.simplex TCCC 11037 to constructed a genetic expression system,and tested it by selecting different hosts to express genes from different sources.Finaly the performance of recombination strains was analyzed.Firstly,using GFP as reporter protein and pART2,a constitutive expression vector,we optimized four parameters of electroporation system of A.simplex to achieve a high electroporation efficiency by monofactorial design.The optimized parameters achieving a electroporation efficiency of 3.76×104 cfu/μg DNA were as follows:cell in the exponential phase(OD600=1.0),a penicillin concentration of 70 μg/mL to cluture the cell for 1 h,200 ng DNA addition mixed with competence cell and the 22 kV/cm-voltage.By using the optimized electroporational parameters,the foreign gene irrE(from Deinococcus Radiodurans R1)was heterologously expressed in different host strains(A.simplex and Arthrobacter globiformis)with pART2 as expression vector.The expression of protein IrrE was verified by HIS purification and Western Blot,which indicated that the expression system mentioned above was applicable in this case.Three expression plasmid named pLJ1、pLJ2 and pLJ3 were constructed on the basis of pART2,which were ligated with gfp and transformed into A.simplex to confirm their functions by checking the existence of transformants on the LA plates and green fluorescent under fluorescence microscope.The result showed that the plasmid pLJl was able to replicate in A.simplex,but unable to express proteins;both of the two plasmids pLJ2 and pLJ3 were unable to replicate in A.simplex.This study stablishes the foundation for further genetic modification and industrial application of A.simplex.