IPTG (isopropyl-β-D-thiogalactoside) yog ib qho analogue ntawm β-galactosidase substrate, uas yog inducible heev. Nyob rau hauv qhov induction ntawm IPTG, tus inducer tuaj yeem tsim ib qho complex nrog cov repressor protein, Yog li ntawd qhov conformation ntawm cov repressor protein hloov pauv, yog li nws tsis tuaj yeem ua ke nrog lub hom phiaj gene, thiab lub hom phiaj gene tau qhia tawm zoo. Yog li yuav tsum txiav txim siab li cas qhov concentration ntawm IPTG thaum lub sijhawm sim? Puas yog qhov loj dua qhov zoo dua?
Ua ntej, cia peb nkag siab txog lub hauv paus ntsiab lus ntawm IPTG induction: E. coli's lactose operon (element) muaj peb lub noob caj noob ces, Z, Y, thiab A, uas encode β-galactosidase, permease, thiab acetyltransferase, raws li. lacZ hydrolyzes lactose rau hauv glucose thiab galactose, lossis rau hauv allo-lactose; lacY tso cai rau lactose nyob rau hauv ib puag ncig kom dhau los ntawm lub cell membrane thiab nkag mus rau hauv lub cell; lacA hloov cov acetyl pawg los ntawm acetyl-CoA mus rau β-galactoside, uas cuam tshuam nrog kev tshem tawm Toxic effect. Tsis tas li ntawd, muaj ib qho kev ua haujlwm sequence O, ib qho pib sequence P thiab ib qho kev tswj hwm gene I. Lub I gene code yog ib qho repressor protein uas tuaj yeem khi rau qhov chaw O ntawm tus neeg teb xov tooj sequence, yog li ntawd operon (meta) raug repressed thiab kaw. Kuj tseem muaj ib qho chaw khi rau catabolic gene activator protein-CAP binding site upstream ntawm qhov pib sequence P. Lub P sequence, O sequence thiab CAP binding site ua ke tsim cov cheeb tsam tswj hwm ntawm lac operon. Cov noob caj noob ces ntawm peb cov enzymes raug tswj hwm los ntawm tib cheeb tsam tswj hwm kom ua tiav kev sib koom tes ntawm cov khoom noob caj noob ces.
Yog tsis muaj lactose, lac operon (meta) nyob rau hauv lub xeev ntawm kev txwv. Lub sijhawm no, lac repressor qhia los ntawm I sequence nyob rau hauv kev tswj hwm ntawm PI promoter sequence khi rau O sequence, uas tiv thaiv RNA polymerase los ntawm kev khi rau P sequence thiab inhibits transcription initiation; thaum lactose muaj, lac operon (meta) tuaj yeem raug induced Hauv no operon (meta) system, tus inducer tiag tiag tsis yog lactose nws tus kheej. Lactose nkag mus rau hauv lub cell thiab yog catalyzed los ntawm β-galactosidase kom hloov mus rau allolactose. Qhov kawg, ua ib tug inducer molecule, khi rau lub repressor protein thiab hloov cov protein conformation, uas ua rau lub dissociation ntawm lub repressor protein los ntawm O sequence thiab transcription. Isopropylthiogalactoside (IPTG) muaj tib yam nyhuv li allolactose. Nws yog ib tug inducer muaj zog heev, uas tsis metabolized los ntawm cov kab mob thiab ruaj khov heev, yog li nws yog dav siv hauv chav kuaj mob.
Yuav ua li cas thiaj paub qhov concentration zoo tshaj plaws ntawm IPTG? Piv txwv li, siv E. coli.
Cov kab mob E. coli BL21 uas tau tsim kho los ntawm kev hloov pauv caj ces uas muaj cov tshuaj pGEX zoo (CGRP/msCT) tau txhaj rau hauv cov kua dej LB uas muaj 50μg·mL-1 Amp, thiab cog qoob loo thaum hmo ntuj ntawm 37°C. Cov kab lis kev cai saum toj no tau txhaj rau hauv 10 lub raj mis ntawm 50mL kua dej LB tshiab uas muaj 50μg·mL-1 Amp ntawm qhov sib piv ntawm 1:100 rau kev nthuav dav kab lis kev cai, thiab thaum tus nqi OD600 yog 0.6 ~ 0.8, IPTG tau ntxiv rau qhov concentration kawg. Nws yog 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mmol·L-1. Tom qab induction ntawm tib qhov kub thiab tib lub sijhawm, 1 mL ntawm cov tshuaj tua kab mob tau coj los ntawm nws, thiab cov hlwb kab mob tau sau los ntawm centrifugation thiab raug rau SDS-PAGE los tshuaj xyuas qhov cuam tshuam ntawm cov IPTG sib txawv ntawm cov protein qhia, thiab tom qab ntawd xaiv IPTG concentration nrog cov protein qhia loj tshaj plaws.
Tom qab kev sim, nws yuav pom tias qhov concentration ntawm IPTG tsis loj li sai tau. Qhov no yog vim IPTG muaj qee yam tshuaj lom rau cov kab mob. Tshaj qhov concentration kuj yuav tua lub cell; thiab feem ntau hais lus, peb vam tias cov protein soluble ntau dua uas tau qhia tawm hauv lub cell, qhov zoo dua, tab sis feem ntau thaum qhov concentration ntawm IPTG siab dhau, ntau qhov kev koom ua ke yuav tsim. Lub cev, tab sis qhov ntau ntawm cov protein soluble txo qis. Yog li ntawd, qhov concentration IPTG tsim nyog tshaj plaws feem ntau tsis yog qhov loj dua qhov zoo dua, tab sis qhov concentration qis dua.
Lub hom phiaj ntawm kev tsim thiab kev cog qoob loo ntawm cov kab mob uas tau tsim kho tshiab yog kom nce cov txiaj ntsig ntawm cov protein uas tau tsim thiab txo cov nqi. Kev qhia tawm ntawm cov noob caj ces tsis yog tsuas yog cuam tshuam los ntawm cov yam ntxwv ntawm cov kab mob thiab cov plasmid uas qhia tawm xwb, tab sis kuj los ntawm lwm yam xwm txheej sab nraud, xws li qhov concentration ntawm tus inducer, qhov kub thiab txias ntawm kev tsim thiab lub sijhawm tsim. Yog li ntawd, feem ntau, ua ntej cov protein tsis paub raug qhia tawm thiab ntxuav, nws yog qhov zoo tshaj plaws los kawm lub sijhawm tsim, qhov kub thiab IPTG concentration kom xaiv cov xwm txheej tsim nyog thiab tau txais cov txiaj ntsig zoo tshaj plaws.
Lub sijhawm tshaj tawm: Lub Kaum Ob Hlis-31-2021