Title: Transforming E. coli and B. subtilis With Plasmid Encoding Gluten-Degrading Enzymes
Jessica (Jia Hui) Wang (Presenter)
Eva Helmerhorst, Boston University
Guoxian Wei, Boston University
Objectives: Celiac disease is characterized by a life-long gluten sensitivity accompanied by an auto-immune reaction. Gluten-degrading enzyme Rmep have been isolated from Rothia mucilaginosa, a natural colonizer of the oral cavity. Like nattokinase, Rmep degrades an immunogenic alpha-gliadin peptide and has important implications as enzyme therapy for celiac disease patients. We aim to initiate transformation of E. coli and B. subtilis with plasmids encoding gluten-degrading enzymes: Rmep isolated from R. mucilaginosa, and nattokinase from B. subtilis.
Methods: Plasmids pHT43-Rm encoding Rmep and pHT01-Bs encoding nattokinase were used for the transformations, with pUC19 as a control. E. coli DE3 and B. subtilis WB800N were used as competent cells. E. coli DE3 cells were first transformed with pUC19 and pHT01-Bs by heat shock. B. subtilis WB800N were transformed with pHT43-Rm, pHT01-Bs and pUC19 by heat shock and by following conventional electroporation protocol. IPTG induction and AAPF cleavage test were conducted to examine endogenous and secreted nattokinase activity in transformants.
Results: Following heat shock using pUC19 and pHT01-Bs, transformation of E. coli was achieved and the transformed cells grew abundantly on LB-Amp plates, although expression was not induced by IPTG and AAPF cleavage activity was not detected. This may be attributed to E. coli’s lack of cellular machinery for protein processing. B. subtilis WB800N subject to both heat shock and electroporation grew on LB plates but did not grow on LB-Amp plates, demonstrating unsuccessful transformation using conventional methods.
Conclusions: E. coli DE3 was successfully transformed with pUC19 and pHT01-Bs by heat shock. Yet, conventional electroporation methods for B. subtilis WB800N require further optimization. Potential modifications include adding trehalose in the electroporation media while applying a stronger pulse.