IPTG (isopropyl-β-D-thiogalactoside) bụ ihe atụ nke β-galactosidase substrate, nke a na-akpali nke ukwuu. N'okpuru mkpali nke IPTG, ihe na-akpali nwere ike ịmepụta ihe mgbagwoju anya na protein repressor, nke mere na agbanweela nhazi nke protein repressor, nke na enweghị ike ijikọ ya na mkpụrụ ndụ ihe nketa ebumnuche, a na-egosipụtakwa mkpụrụ ndụ ihe nketa ebumnuche nke ọma. Yabụ kedu ka esi achọpụta ntinye nke IPTG n'oge nnwale ahụ? Ò buru ibu ka mma?
Nke mbụ, ka anyị ghọta ụkpụrụ nke IPTG induction: lactose operon (ihe) nke E. coli nwere mkpụrụ ndụ ihe nketa atọ, Z, Y, na A, nke na-akọwapụta β-galactosidase, permease, na acetyltransferase, n'otu n'otu. lacZ na-eme ka lactose ghọọ glucose na galactose, ma ọ bụ banye na allo-lactose; lacY na-ekwe ka lactose dị na gburugburu ebe obibi gafee site na akpụkpọ ahụ sel wee banye na sel; lacA na-ebufe otu acetyl site na acetyl-CoA gaa na β-galactoside, nke gụnyere iwepụ mmetụta Toxic. Na mgbakwunye, enwere usoro ọrụ O, usoro mmalite P na mkpụrụ ndụ ihe nketa I. Koodu mkpụrụ ndụ ihe nketa I bụ protein repressor nke nwere ike ijikọ na ọnọdụ O nke usoro onye ọrụ, ka o wee gbochie ma gbanyụọ operon (meta). E nwekwara ebe njikọ maka ebe njikọ protein-CAP nke na-akwalite mkpụrụ ndụ ihe nketa catabolic nke usoro mmalite P. Usoro P, usoro O na ebe njikọ CAP jikọtara ọnụ bụ mpaghara nchịkwa nke lac operon. A na-achịkwa mkpụrụ ndụ ihe nketa koodu nke enzymes atọ ahụ site na otu mpaghara nchịkwa iji nweta ngosipụta nhazi nke ngwaahịa mkpụrụ ndụ ihe nketa.
Ọ bụrụ na lactose adịghị, lac operon (meta) nọ n'ọnọdụ nrụgide. N'oge a, lac repressor nke usoro I gosipụtara n'okpuru njikwa usoro nkwalite PI na-ejikọ na usoro O, nke na-egbochi RNA polymerase ijikọ na usoro P ma na-egbochi mmalite ntụgharị; mgbe lactose dị, enwere ike ịkpalite lac operon (meta) N'ime sistemụ operon (meta) a, ezigbo ihe na-akpali abụghị lactose n'onwe ya. Lactose na-abanye na sel ma β-galactosidase na-akpali ya ka ọ bụrụ allolactose. Nke ikpeazụ, dị ka molekul inducer, na-ejikọ na protein repressor ma na-agbanwe nhazi protein, nke na-eduga na nkewa nke protein repressor site na usoro O na transcription. Isopropylthiogalactoside (IPTG) nwere otu mmetụta ahụ dị ka allolactose. Ọ bụ ihe na-akpali akpali dị ike, nke nje bacteria na-anaghị eme ka ọ dị nro ma dịkwa mma, yabụ a na-ejikarị ya eme ihe n'ụlọ nyocha.
Kedu otu esi achọpụta oke IPTG dị? Were E. coli dịka ọmụmaatụ.
E tinyere ọgwụ nje E. coli BL21 nke nwere pGEX recombinant dị mma (CGRP/msCT) n'ime mmiri mmiri LB nke nwere 50μg·mL-1 Amp, ma kụọ ya n'abalị na 37°C. E tinyere ọgwụ nje a n'elu n'ime karama iri nke mmiri mmiri 50mL ọhụrụ nke nwere 50μg·mL-1 Amp na nhata nke 1:100 maka ọdịbendị mgbasa, mgbe uru OD600 bụ 0.6~0.8, etinyere IPTG na mkpokọta ikpeazụ. Ọ bụ 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mmol·L-1. Mgbe e tinyere ya n'otu okpomọkụ ahụ na otu oge ahụ, e si na ya wepụta 1 mL nke ihe ngwọta nje bacteria, a na-anakọtakwa mkpụrụ ndụ nje bacteria site na centrifugation ma tinye SDS-PAGE iji nyochaa mmetụta nke mkpokọta IPTG dị iche iche na ngosipụta protein, wee họrọ mkpokọta IPTG nwere ngosipụta protein kachasị.
Mgbe emechara nnwale, a ga-achọpụta na ntinye IPTG adịghị oke oke o kwere mee. Nke a bụ n'ihi na IPTG nwere ụfọdụ nsí na nje bacteria. Ịgabiga ntinye ahụ ga-egbukwa mkpụrụ ndụ ahụ; n'ozuzu, anyị na-atụ anya na protein na-agbaze nke ọma na mkpụrụ ndụ ahụ, ka mma, mana n'ọtụtụ oge mgbe ntinye IPTG dị oke elu, a ga-emepụta nnukwu ntinye. Ahụ, mana ọnụọgụ protein na-agbaze belatara. Ya mere, ntinye IPTG kacha mma anaghị abụkarị ibu ka mma, kama obere ntinye ahụ.
Ebumnuche nke ịmalite na ịzụlite ụdị mkpụrụ ndụ ihe nketa bụ ịbawanye mmepụta nke protein ebumnuche ma belata ọnụ ahịa. Ngosipụta nke mkpụrụ ndụ ihe nketa ebumnuche abụghị naanị site na ihe ndị metụtara nje ahụ na ngosipụta plasmid, kamakwa site na ọnọdụ mpụga ndị ọzọ, dịka ntinye nke ihe mkpali, okpomọkụ induction na oge induction. Ya mere, n'ozuzu, tupu egosipụta ma sachaa protein a na-amaghị ama, ọ kacha mma ịmụ oge induction, okpomọkụ na ntinye IPTG iji họrọ ọnọdụ kwesịrị ekwesị ma nweta nsonaazụ nnwale kacha mma.
Oge ozi: Disemba-31-2021