I-IPTG (isopropyl-β-D-thiogalactoside) yi-analogue ye-β-galactosidase substrate, engakwazi ukunyibilika kakhulu. Phantsi kokungeniswa kwe-IPTG, i-inducer inokwenza i-complex ene-repressor protein, ukuze i-conformation ye-repressor protein itshintshe, ukuze ingakwazi ukudityaniswa ne-target gene, kwaye i-target gene ibonakaliswe ngokufanelekileyo. Ngoko ke kufuneka kumiselwe njani ukuxinana kwe-IPTG ngexesha lovavanyo? Ngaba inkulu ingcono?
Okokuqala, masiqonde umgaqo wokungeniswa kwe-IPTG: I-lactose operon (into) ye-E. coli ineejini ezintathu zesakhiwo, i-Z, i-Y, kunye ne-A, ezifaka i-β-galactosidase, i-permease, kunye ne-acetyltransferase, ngokulandelelana. I-lacZ i-hydrolyzes i-lactose ibe yi-glucose kunye ne-galactose, okanye ibe yi-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, equka ukususa isiphumo seTyhefu. Ukongeza, kukho ulandelelwano olusebenzayo lwe-O, ulandelelwano lokuqala lwe-P kunye ne-gene elawulayo ye-I. Ikhowudi ye-gene ye-I yiproteni ecinezelayo enokubopha kwindawo ye-O ye-operator sequence, ukuze i-operon (i-meta) icinezelwe kwaye icinywe. Kukwakho nendawo yokubopha ye-catabolic gene activator protein-CAP binding site phezulu kwe-initiative sequence P. I-P sequence, i-O sequence kunye ne-CAP binding site kunye zenza ummandla olawulayo we-lac operon. Iijini zekhowudi zee-enzymes ezintathu zilawulwa yindawo efanayo yokulawula ukuze kufezekiswe ukubonakaliswa okudibeneyo kweemveliso zejini.
Xa kungekho lactose, i-lac operon (meta) ikwimeko yokucinezelwa. Ngeli xesha, i-lac repressor ebonakaliswa lulandelelwano lwe-I phantsi kolawulo lwe-PI promoter sequence ibopha kwi-O sequence, ethintela i-RNA polymerase ekubeni ibophane kwi-P sequence kwaye ithintele ukuqaliswa kwe-transcription; xa i-lactose ikhona, i-lac operon (meta) inokubangelwa Kule nkqubo ye-operon (meta), i-inducer yokwenyani ayisiyo lactose ngokwayo. I-Lactose ingena kwiseli kwaye i-catalyzed yi-β-galactosidase ukuze iguqulwe ibe yi-allolactose. Le yokugqibela, njengemolekyuli ye-inducer, ibopha kwiproteni ye-repressor kwaye itshintsha ukwakheka kweproteni, okukhokelela ekuqhekekeni kweproteni ye-repressor kwi-O sequence kunye ne-transcription. I-Isopropylthiogalactoside (IPTG) inefuthe elifanayo ne-allolactose. Yi-inducer enamandla kakhulu, engaxutywanga yibhaktheriya kwaye izinzile kakhulu, ngoko ke isetyenziswa kakhulu kwiilabhoratri.
Ungayifumanisa njani i-IPTG efanelekileyo? Umzekelo, cinga nge-E. coli.
Uhlobo lwe-E. coli BL21 oluyilwe ngokwezakhi zofuzo oluqulethe i-positive recombinant pGEX (CGRP/msCT) lufakwe kwi-LB liquid medium equlethe i-50μg·mL-1 Amp, kwaye lukhuliswe ubusuku bonke kwi-37°C. Olu hlobo lungentla lufakwe kwiibhotile ezili-10 ze-50mL fresh LB liquid medium equlethe i-50μg·mL-1 Amp ngomlinganiselo we-1:100 wokukhulisa, kwaye xa ixabiso le-OD600 lalingu-0.6~0.8, i-IPTG yongezwa kuxinzelelo lokugqibela. Yi-0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mmol·L-1. Emva kokungeniswa kubushushu obufanayo nangexesha elifanayo, i-1 mL yesisombululo sebhaktiriya yathathwa kuyo, kwaye iiseli zebhaktiriya zaqokelelwa nge-centrifugation zaza zafakwa kwi-SDS-PAGE ukuze kuhlalutywe impembelelo yamanqanaba ahlukeneyo e-IPTG ekubonakalisweni kweproteni, emva koko kukhethwe ugxininiso lwe-IPTG olune-protein expression enkulu.
Emva kovavanyo, kuya kufunyaniswa ukuba uxinaniso lwe-IPTG alukho mkhulu kangangoko. Oku kungenxa yokuba i-IPTG inetyhefu ethile kwiibhaktheriya. Ukudlula uxinaniso kuya kubulala iseli; kwaye ngokubanzi, sinethemba lokuba iproteni enyibilikayo ngakumbi evezwa kwiseli, kokukhona ingcono, kodwa kwiimeko ezininzi xa uxinaniso lwe-IPTG luphezulu kakhulu, kuya kubakho inani elikhulu lokufakwa. Umzimba, kodwa ubungakanani beproteni enyibilikayo buyancipha. Ke ngoko, uxinaniso olufanelekileyo lwe-IPTG ludla ngokuba lungabi lukhulu, kodwa luphantsi uxinaniso.
Injongo yokungenisa nokukhulisa iintlobo eziguqulwe ngokwezakhi zofuzo kukunyusa isivuno seprotheyini ekujoliswe kuyo kunye nokunciphisa iindleko. Ukubonakaliswa kwe-gene ekujoliswe kuyo akuchatshazelwa kuphela zizinto zohlobo kunye ne-plasmid yokubonakaliswa, kodwa nakwezinye iimeko zangaphandle, ezifana noxinzelelo lwe-inducer, ubushushu bokungeniswa kunye nexesha lokungeniswa. Ke ngoko, ngokubanzi, ngaphambi kokuba iprotheyini engaziwayo ivezwe kwaye icocwe, kungcono ukufunda ixesha lokungeniswa, ubushushu kunye noxinzelelo lwe-IPTG ukuze ukhethe iimeko ezifanelekileyo kwaye ufumane iziphumo ezilungileyo zovavanyo.
Ixesha lokuthumela: Disemba-31-2021