I-IPTG (isopropyl-β-D-thiogalactoside) iyi-analogue ye-β-galactosidase substrate, ekwazi ukungena kalula.Ngaphansi kokungeniswa kwe-IPTG, i-inducer ingenza inkimbinkimbi neprotheyini yokucindezela, Ukuze ukuguqulwa kweprotheni yokucindezela kuguqulwe, ukuze kungakwazi ukuhlanganiswa nesakhi esihlosiwe, futhi isakhi sofuzo esihlosiwe siboniswa ngokuphumelelayo.Ngakho-ke ukugxila kwe-IPTG kufanele kunqunywe kanjani phakathi nokuhlolwa?Ingabe enkulu iyona engcono?
Okokuqala, masiqonde isimiso sokungeniswa kwe-IPTG: I-E. coli's lactose operon (elementi) iqukethe izakhi zofuzo ezintathu zesakhiwo, Z,Y, kanye no-A, ezihlanganisa i-β-galactosidase, permease, ne-acetyltransferase, ngokulandelana.i-lacZ i-hydrolyze i-lactose ibe yi-glucose ne-galactose, noma ibe yi-allo-lactose;i-lacY ivumela i-lactose endaweni ukuthi idlule kulwelwesi lweseli futhi ingene esitokisini;I-lacA idlulisela iqembu le-acetyl lisuka ku-acetyl-CoA liye ku-β-galactoside, okubandakanya ukususa umphumela Onobuthi.Ukwengeza, kukhona ukulandelana kokusebenza kwe-O, ukulandelana kokuqala kwe-P kanye ne-gene elawulayo I. Ikhodi yofuzo ye-I iyiphrotheni yokucindezela engakwazi ukubophezela esikhundleni se-O sokulandelana komsebenzisi, ukuze i-operon (meta) icindezelwe futhi. icishiwe.Kukhona futhi indawo ebophezelayo yesayithi ehlanganisa i-catabolic gene activator protein-CAP enhla nomfula wokulandelana kokuqala P. Ukulandelana kwe-P, uchungechunge lwe-O kanye nesayithi elihlanganisayo le-CAP ndawonye lakha indawo yokulawula ye-lac operon.Izakhi zofuzo zekhodi zama-enzyme amathathu zilawulwa yisifunda sokulawula esifanayo ukuze kuzuzwe ukuvezwa okuhlanganisiwe kwemikhiqizo yofuzo.
Uma ingekho i-lactose, i-lac operon (meta) isesimweni sokucindezelwa.Ngalesi sikhathi, i-lac repressor evezwe ukulandelana kwe-I ngaphansi kokulawulwa kokulandelana komgqugquzeli we-PI ibophezela ukulandelana kwe-O, okuvimbela i-RNA polymerase ekubopheni ukulandelana kuka-P futhi ivimbele ukuqaliswa kokubhala;uma i-lactose ikhona, i-lac operon (i-meta) ingenziwa kulolu hlelo lwe-operon (meta), i-inducer yangempela ayiyona i-lactose ngokwayo.I-Lactose ingena engqamuzaneni futhi ikhiqizwa yi-β-galactosidase ukuze iguqulelwe ku-alllolactose.Lesi sakamuva, njenge-molecule ye-inducer, ibophezela kuphrotheni yokucindezela futhi iguqule ukuguqulwa kwamaprotheni, okuholela ekuhlukaniseni iphrotheni yokucindezela kusukela ekulandeleni kwe-O nokubhala.I-Isopropylthiogalactoside (IPTG) inomphumela ofanayo ne-alllolactose.Kuyi-inducer enamandla kakhulu, engenziwanga i-metabolized by bacteria futhi izinzile kakhulu, ngakho-ke isetshenziswa kabanzi kuma-laboratories.
Unganquma kanjani ukugxiliswa okuphelele kwe-IPTG?Thatha i-E. coli njengesibonelo.
I-E. coli BL21 uhlobo olwenziwe ngofuzo oluqukethe i-positive recombinant pGEX (CGRP/msCT) yafakwa ku-LB liquid medium equkethe 50μg·mL-1 Amp, futhi yakhuliswa ngobusuku obubodwa ku-37°C.Isiko elingenhla lifakwe emabhodleleni angu-10 we-50mL fresh LB liquid medium aqukethe 50μg·mL-1 Amp ngesilinganiso esingu-1:100 sokwandisa isiko, futhi lapho inani le-OD600 lingu-0.6~0.8, i-IPTG yengezwa ekugxiliseni kokugcina.Kungu-0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mmol·L-1.Ngemuva kokungeniswa ezingeni lokushisa elifanayo nangesikhathi esifanayo, i-1 mL yesisombululo sebhaktheriya yathathwa kuyo, futhi amangqamuzana ebhaktheriya aqoqwa nge-centrifugation futhi afakwa ngaphansi kwe-SDS-PAGE ukuze ahlaziye umthelela wokugxila okuhlukile kwe-IPTG ekukhulumeni kwamaprotheni, bese-ke. khetha ukugxiliswa kwe-IPTG ngesisho esikhulu samaprotheni.
Ngemuva kokuhlolwa, kuzotholakala ukuthi ukugxila kwe-IPTG akukukhulu ngangokunokwenzeka.Lokhu kungenxa yokuthi i-IPTG inobuthi obuthile kubhaktheriya.Ukweqa ukugxila kuzophinde kubulale ingqamuzana;futhi ngokujwayelekile, sithemba ukuthi iphrotheni encibilikayo evezwe kuseli, ingcono, kodwa ezimweni eziningi lapho ukugxila kwe-IPTG kuphezulu kakhulu, inani elikhulu lokufakwa lizokwakhiwa.Umzimba, kodwa inani lamaprotheni ancibilikayo liyehla.Ngakho-ke, ukugxiliswa kwe-IPTG okufanele kakhulu ngokuvamile akubi kakhulu kangcono, kodwa kuyancipha ukugxila.
Inhloso yokwethulwa kanye nokutshalwa kwezinhlobo zofuzo ukukhulisa isivuno samaprotheni okuhlosiwe kanye nokunciphisa izindleko.Ukuvezwa kofuzo oluqondiwe akuthintwa nje kuphela izici zohlobo oluthile kanye nenkulumo ethi plasmid, kodwa futhi nangezinye izimo zangaphandle, njengokugxilwa kwe-inducer, izinga lokushisa lokungeniswa kanye nesikhathi sokungeniswa.Ngakho-ke, ngokuvamile, ngaphambi kokuba iphrotheni engaziwa ivezwe futhi ihlanzwe, kungcono ukutadisha isikhathi sokungeniswa, izinga lokushisa kanye nokugxila kwe-IPTG ukuze ukhethe izimo ezifanele futhi uthole imiphumela engcono kakhulu yokuhlola.
Isikhathi sokuthumela: Dec-31-2021