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I-IPTG (isopropyl-β-D-thiogalactoside) i-analogue ye-β-galactosidase substrate, enokuthi i-inducible kakhulu.Ngaphantsi kokungeniswa kwe-IPTG, i-inducer inokwenza i-complex kunye neprotheyini ye-repressor , Ukuze ukuguqulwa kweprotheni ye-repressor iguqulwe, ukwenzela ukuba ingenakudibaniswa ne-gene ekujoliswe kuyo, kwaye i-gene ekujoliswe kuyo ibonakaliswe ngokufanelekileyo.Ngoko ke kufuneka kumiselwe njani ugxininiso lwe-IPTG ngexesha lovavanyo?Ngaba inkulu ingcono?
Okokuqala, masiqonde umgaqo we-IPTG wokufakwa: I-E. coli's lactose operon (i-element) iqulethe iijene ezintathu zesakhiwo, i-Z,Y, kunye ne-A, edibanisa i-β-galactosidase, i-permease, kunye ne-acetyltransferase, ngokulandelanayo.i-lacZ i-hydrolyze i-lactose kwi-glucose kunye ne-galactose, okanye kwi-allo-lactose;I-lacY ivumela i-lactose kwindalo ukuba idlule kwi-membrane yeseli kwaye ingene kwiseli;I-lacA idlulisela iqela le-acetyl ukusuka kwi-acetyl-CoA ukuya kwi-β-galactoside, ebandakanya ukususa umphumo we-Toxic.Ukongezelela, kukho ukulandelelana kokusebenza kwe-O, ukulandelelana kokuqala kwe-P kunye ne-gene yokulawula i-I. Ikhowudi ye-gene ye-gene iyiprotheni ye-repressor enokuthi ibophe kwindawo ye-O yokulandelelana komsebenzi, ukwenzela ukuba i-operon (meta) igxininiswe kwaye icinyiwe.Kukwakho nesiza esibophelelayo sesiza secatabolic gene activator protein-CAP binding site upstream of the initiating sequence P. I-P yolandelelwano, i-O yolandelelwano kunye ne-CAP edibanisa indawo kunye zenza ummandla wolawulo we-lac operon.Iijene zekhowudi zee-enzyme ezintathu zilawulwa ngummandla wolawulo ofanayo ukuze kuphunyezwe ukubonakaliswa okuhambelanayo kweemveliso zofuzo.

Xa kungekho lactose, i-lac operon (meta) ikwimeko yokucinezelwa.Ngeli xesha, i-lac repressor echazwe ngokulandelelana kwe-I phantsi kolawulo lwe-PI umgqugquzeli wokulandelelana ubophelela ku-O ulandelelwano, oluthintela i-RNA polymerase ukuba ibophe ukulandelelana kwe-P kwaye inqanda ukuqaliswa kokubhaliweyo;xa i-lactose ikhona, i-lac operon (meta) inokunyanzeliswa Kule nkqubo ye-operon (meta), i-inducer yangempela ayikho i-lactose ngokwayo.I-Lactose ingena kwiseli kwaye i-catalyzed yi-β-galactosidase ukuze iguqulwe ibe yi-alllolactose.Le yokugqibela, njenge-molecule ye-inducer, ibophelela kwiprotheni ye-repressor kwaye iguqule i-protein conformation, ekhokelela ekuhlukaneni kweprotheni ye-repressor ukusuka kwi-O yokulandelelana kunye nokubhala.I-Isopropylthiogalactoside (IPTG) inefuthe elifanayo ne-alllolactose.I-inducer enamandla kakhulu, engenziwanga i-metabolized yi-bacteria kwaye izinzile kakhulu, ngoko isetyenziswa ngokubanzi kwiibhubhoratri.

Indlela yokumisela eyona ngxinaniso ye-IPTG?Thatha i-E. coli njengomzekelo.
I-E. coli BL21 yohlobo lwe-genetically engineered strain equkethe i-pGEX ehlaziyiweyo (CGRP/msCT) yagonywa kwi-LB medium medium equlethe i-50μg · mL-1 Amp, kwaye ikhuliswe ngobusuku kwi-37 ° C.Inkcubeko engentla ifakwe kwiibhotile ze-10 ze-50mL entsha ye-LB medium medium equkethe i-50μg · mL-1 Amp kwi-1: 100 yenkcubeko yokwandisa, kwaye xa ixabiso le-OD600 laliyi-0.6 ~ 0.8, i-IPTG yongezwa kwi-concentration yokugqibela.Ngu-0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mmol · L-1.Emva kokungeniswa kwiqondo lokushisa elifanayo kunye nexesha elifanayo, i-1 mL yesisombululo sebhaktheriya ithathwe kuyo, kwaye iiseli zebhaktheriya zaqokelelwa nge-centrifugation kwaye ziphantsi kwe-SDS-PAGE ukuhlalutya impembelelo ye-IPTG yokugxininiswa kweeprotheni ezahlukeneyo, emva koko. khetha i-IPTG yoxinaniso eneprotheyini enkulu yokubonakalisa.
Emva kovavanyo, kuya kufunyaniswa ukuba ukuxinana kwe-IPTG akukhulu kangangoko kunokwenzeka.Oku kungenxa yokuba i-IPTG inetyhefu ethile kwibhaktheriya.Ukugqithisa ugxininiso kuya kubulala iseli;kwaye ngokuqhelekileyo ukuthetha, sinethemba lokuba iprotheni enyibilikayo echazwe kwiseli, ingcono, kodwa kwiimeko ezininzi xa uxinaniso lwe-IPTG luphezulu kakhulu, inani elikhulu lokubandakanywa liya kwenziwa.Umzimba, kodwa isixa seprotheyini enyibilikayo sehlile.Ke ngoko, eyona nto ifanelekileyo yoxinaniso ye-IPTG ihlala ingabi nkulu ngakumbi, kodwa iphantsi koxinaniso.
Injongo yokufakwa kunye nokulinywa kweentlobo ze-genetically engineered strains kukunyusa isivuno seprotheyini ekujoliswe kuyo kunye nokunciphisa iindleko.Ukubonakaliswa kwejini ekujoliswe kuyo akuchatshazelwa kuphela yimiba ye-strain kunye ne-plasmid ye-expression, kodwa kunye nezinye iimeko zangaphandle, ezifana nokugxininiswa kwe-inducer, ukushisa kwe-induction kunye nexesha lokungeniswa.Ngoko ke, ngokuqhelekileyo, ngaphambi kokuba iprotheni engaziwayo ibonakaliswe kwaye ihlanjululwe, kukulungele ukufunda ixesha lokungeniswa, ukushisa kunye noxinzelelo lwe-IPTG ukwenzela ukuba ukhethe iimeko ezifanelekileyo kwaye ufumane iziphumo ezilungileyo zokuvavanya.