ozi

IPTG (isopropyl-β-D-thiogalactoside) bụ ihe analọg nke mkpụrụ β-galactosidase, nke na-enweghị ike ime ya nke ukwuu.N'okpuru ntinye nke IPTG, onye na-emepụta ihe nwere ike ịmepụta mgbagwoju anya na protein repressor , Ka e wee gbanwee ngbanwe nke protein repressor, nke mere na enweghi ike ijikọta ya na mkpụrụ ndụ ihe mgbaru ọsọ, na-egosipụtakwa mkpụrụ ndụ ihe nke ọma.Yabụ kedu ka esi ekpebi itinye uche nke IPTG n'oge nnwale ahụ?Nke ka ukwuu ọ ka mma?
Nke mbụ, ka anyị ghọta ụkpụrụ nke induction IPTG: E. coli's lactose operon (ihe) nwere mkpụrụ ndụ ihe nrụzi atọ, Z,Y, na A, nke na-edekọ β-galactosidase, permease, na acetyltransferase, n'otu n'otu.lacZ hydrolyzes lactose n'ime glucose na galactose, ma ọ bụ n'ime allo-lactose;lacY na-enye ohere ka lactose dị na gburugburu ebe obibi gafere site na cell membrane wee banye na cell;lacA na-ebufe otu acetyl site na acetyl-CoA gaa na β-galactoside, nke gụnyere iwepụ mmetụta nsị.Tụkwasị na nke ahụ, e nwere usoro ọrụ O, usoro mmalite P na usoro nhazi usoro I. I gene code bụ protein repressor nke nwere ike jikọta na ọnọdụ O nke usoro onye na-arụ ọrụ, nke mere na operon (meta) na-emegharị na. gbanyụọ.Enwekwara saịtị njikọ maka catabolic gene activator protein-CAP njikọ elu nke usoro mmalite P. Usoro P, usoro O na saịtị CAP jikọtara ọnụ mejupụtara mpaghara nchịkwa nke lac operon.A na-ahazi mkpụrụ ndụ ihe nketa nke enzymes atọ ahụ site n'otu mpaghara na-achịkwa iji nweta nkwupụta ahaziri ahazi nke ngwaahịa mkpụrụ ndụ ihe nketa.

Na enweghị lactose, lac operon (meta) nọ n'ọnọdụ mmegbu.N'oge a, lac repressor gosipụtara site na usoro nke I n'okpuru nchịkwa nke usoro mgbasa ozi PI na-ejikọta ya na usoro O, nke na-egbochi RNA polymerase ka ọ ghara ijikọta ya na usoro P ma gbochie mmalite nsụgharị;mgbe lactose dị, enwere ike ịmalite lac operon (meta) Na usoro operon (meta), ezigbo inducer abụghị lactose n'onwe ya.Lactose na-abanye n'ime cell ma na-eme ka β-galactosidase gbanwee ka ọ ghọọ allolactose.Nke ikpeazụ, dị ka molecule inducer, na-ejikọta na protein repressor ma gbanwee nhazi protein, nke na-eduga na nkwụsị nke protein repressor site na usoro O na ntụgharị.Isopropylthiogalactoside (IPTG) nwere otu mmetụta dị ka allolactose.Ọ bụ ihe na-eme ka ọ dị ike nke ukwuu, nke nje bacteria na-adịghị edozi ya ma kwụsie ike nke ukwuu, ya mere a na-eji ya na ụlọ nyocha.

Kedu otu esi achọpụta mkpokọta kacha mma nke IPTG?Were E. coli dị ka ihe atụ.
E. coli BL21 a na-emepụta mkpụrụ ndụ ihe nketa nke nwere ezi recombinant pGEX (CGRP/msCT) ka etinyere n'ime mmiri mmiri LB nke nwere 50μg·mL-1 Amp, ma zụlite ya n'otu abalị na 37°C.A na-etinye omenala ndị dị n'elu n'ime karama 10 nke 50mL ọhụrụ LB mmiri mmiri nwere 50μg·mL-1 Amp na nha nke 1: 100 maka ọdịbendị mgbasawanye, na mgbe OD600 uru bụ 0.6 ~ 0.8, agbakwunyere IPTG na ntinye 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 ntinye n'otu okpomọkụ na n'otu oge ahụ, a na-ewepụ 1 mL nke ngwọta nje na ya, a na-anakọta mkpụrụ ndụ nje site na centrifugation ma tinye ya na SDS-PAGE iji nyochaa mmetụta dị iche iche nke IPTG dị iche iche na nkwupụta protein, na mgbe ahụ. họrọ ntinye uche IPTG nwere okwu protein kacha ukwuu.
Mgbe nyochachara, a ga-achọpụta na ntinye nke IPTG adịghị oke dịka o kwere mee.Nke a bụ n'ihi na IPTG nwere ụfọdụ nsí na nje bacteria.Ịgafe uche ga-egbukwa cell;na n'ikwu okwu n'ozuzu, anyị na-atụ anya na protein na-esi ike na-egosipụta na cell, nke ka mma, ma n'ọtụtụ ọnọdụ mgbe ntinye nke IPTG dị oke elu, a ga-emepụta nnukwu ego nke nsonye.Ahụ, mana ọnụọgụ protein soluble belatara.Ya mere, ntinye uche IPTG kachasị mma anaghị abụkarị nke ka mma, mana ọ na-ebelata ntinye uche.
Ebumnuche nke iwebata na ịkụ mkpụrụ nke ụdị mkpụrụ ndụ ihe nketa na-emepụta bụ ịbawanye mkpụrụ nke protein ezubere iche wee belata ọnụ ahịa.Okwu nke mkpụrụ ndụ ihe nlebara anya abụghị naanị ihe kpatara nje ahụ na okwu plasmid, kamakwa site na ọnọdụ mpụga ndị ọzọ, dị ka ntinye nke inducer, okpomọkụ induction na oge ntinye.Ya mere, n'ozuzu, tupu e gosipụta protein na-amaghị ama ma dị ọcha, ọ kacha mma ka ị mụọ oge ntinye, okpomọkụ na ntinye uche IPTG iji họrọ ọnọdụ kwesịrị ekwesị ma nweta nsonaazụ nnwale kacha mma.