AgreatamountoffoodbornepathogenswereGram-positive(G?)bacteria,athreattopublichealth.Inthisstudy,consideringthebindingabilityofnisintowardsG-bacteriaandthestablefluorescentabilityofEGFPprotein,afluorescentnisin–EGFPproteinprobewasconstructedbyageneengineeringmethod.NisinandEGFPwereusedasthereceptorandfluorophore,respectively,todetectG?bacteria.ThenisinandegfpgenewereamplifiedseparatelyaccordingtothesequencepublishedinGenBankusinguniqueprimers.ThetwogeneswereclonedintoapET-28b(?)vectorresultinginapET-28b(?)–nisin–egfpvector.ThevectorwastransferredintoEscherichiacoli(E.coli)BL21(DE3)forexpression.Theexpressedproteinwasextracted,purifiedbyaNi–NTAcolumn,andthentestedbytheSDS-PAGEmethodtoconfirmitsmolecularweight.Listeriamonocytogenes(L.monocytogenes),Staphylococcusaureus(S.aureus),andMicrococcusluteus(M.luteus)wereusedastherepresentationsofG?bacteria.E.coliO157,representingthegram-negative(G-)bacteria,wasusedasanegativecontrol.Thebindingspecificityoftherecombinantproteinwasperformedontwotypesofbacteriaandthendetectedthroughfluorescentmicroscopy.Theresultsindicatedthatthenisin–EGFPprobecoulddetectG?bacteriaat108CFU/mL.
