xov xwm

IPTG (isopropyl-β-D-thiogalactoside) yog ib qho analogue ntawm β-galactosidase substrate, uas yog inducible heev.Raws li qhov induction ntawm IPTG, tus inducer tuaj yeem tsim ib txoj hauv kev nrog cov protein repressor, kom cov kev hloov pauv ntawm cov protein repressor hloov, kom nws tsis tuaj yeem ua ke nrog cov hom phiaj, thiab cov hom phiaj tau nthuav tawm zoo.Yog li cas yuav tsum tau txiav txim siab qhov concentration ntawm IPTG thaum lub sijhawm sim?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 yam genes, Z, Y, thiab A, uas encode β-galactosidase, permease, thiab acetyltransferase, feem.lacZ hydrolyzes lactose rau hauv qabzib thiab galactose, lossis rau hauv allo-lactose;lacY tso cai rau lactose nyob rau hauv ib puag ncig kom dhau los ntawm 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 rau kev tshem tawm cov nyhuv Toxic.Tsis tas li ntawd, muaj kev ua haujlwm ua ntu zus O, pib ua ntu P thiab kev tswj hwm gene I. I gene code yog cov protein repressor uas tuaj yeem khi rau txoj haujlwm O ntawm tus neeg teb xov tooj ib ntus, kom lub operon (meta) yog repressed thiab muab tua.Kuj tseem muaj qhov chaw sib txuas rau catabolic gene activator protein-CAP khi qhov chaw nce toj ntawm qhov pib ua ntu zus P. Qhov P ib ntus, O ib ntus thiab CAP khi qhov chaw ua ke ua rau thaj tsam tswj hwm ntawm lac operon.Lub coding noob ntawm peb enzymes yog tswj los ntawm tib cheeb tsam tswj kom ua tiav cov kev sib koom ua ke ntawm cov khoom noob.

Thaum tsis muaj lactose, lac operon (meta) yog nyob rau hauv lub xeev ntawm kev tsim txom.Lub sijhawm no, lac repressor qhia los ntawm I ib ntus raws li kev tswj hwm ntawm PI txhawb nqa ib ntus khi rau O sequence, uas tiv thaiv RNA polymerase los ntawm kev khi rau P ib ntus thiab inhibits kev pib transcription;Thaum lactose tshwm sim, lac operon (meta) tuaj yeem raug ntxias Hauv qhov system operon (meta), qhov tseeb inducer tsis yog lactose nws tus kheej.Lactose nkag mus rau hauv lub cell thiab catalyzed los ntawm β-galactosidase kom hloov mus rau allolactose.Lub tom kawg, raws li ib tug inducer molecule, khi rau repressor protein thiab hloov cov protein conformation, uas ua rau lub dissociation ntawm repressor protein los ntawm lub O sequence thiab transcription.Isopropylthiogalactoside (IPTG) muaj cov nyhuv tib yam li allolactose.Nws yog ib qho inducer muaj zog heev, uas tsis yog metabolized los ntawm cov kab mob thiab ruaj khov heev, yog li nws tau siv dav hauv cov chaw sim.

Yuav ua li cas txiav txim siab qhov zoo tshaj plaws concentration ntawm IPTG?Siv E. coli ua piv txwv.
E. coli BL21 genetically engineered strain uas muaj qhov zoo recombinant pGEX (CGRP/msCT) tau inoculated rau hauv LB kua nruab nrab uas muaj 50μg·mL-1 Amp, thiab cultured hmo ntuj ntawm 37 ° C.Cov kab lis kev cai saum toj no tau muab tso rau hauv 10 fwj ntawm 50mL tshiab LB kua nruab nrab uas muaj 50μg·mL-1 Amp ntawm qhov piv ntawm 1:100 rau kev nthuav dav kab lis kev cai, thiab thaum OD600 tus nqi yog 0.6 ~ 0.8, IPTG tau ntxiv rau qhov kawg concentration.0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mmol · L-1.Tom qab induction ntawm tib qhov kub thiab tib lub sijhawm, 1 mL ntawm cov kab mob tshuaj tau muab los ntawm nws, thiab cov kab mob hlwb tau sau los ntawm centrifugation thiab raug rau SDS-PAGE los tshuaj xyuas qhov cuam tshuam ntawm IPTG sib txawv ntawm cov protein qhia, thiab tom qab ntawd xaiv qhov IPTG concentration nrog cov protein ntau tshaj plaws.
Tom qab kev sim, nws yuav pom tias qhov concentration ntawm IPTG tsis loj npaum li qhov ua tau.Qhov no yog vim IPTG muaj qee yam tshuaj lom rau cov kab mob.Tshaj qhov concentration tseem yuav tua cov cell;thiab feem ntau hais lus, peb cia siab tias cov protein ntau soluble qhia nyob rau hauv lub cell, qhov zoo dua, tab sis nyob rau hauv ntau lub sij hawm thaum lub concentration ntawm IPTG siab dhau lawm, ib tug loj npaum li cas ntawm kev koom tes yuav tsim.Lub cev, tab sis tus nqi ntawm soluble protein tsawg.Yog li ntawd, qhov zoo tshaj plaws IPTG concentration feem ntau tsis yog qhov loj dua qhov zoo dua, tab sis qhov qis dua qhov concentration.
Lub hom phiaj ntawm induction thiab cultivation ntawm genetic engineered strains yog ua kom cov yield ntawm lub hom phiaj protein thiab txo nqi.Kev qhia ntawm lub hom phiaj noob tsis yog tsuas yog cuam tshuam los ntawm lub hom phiaj ntawm tus kheej thiab cov lus qhia plasmid, tab sis kuj los ntawm lwm yam kev mob sab nraud, xws li cov concentration ntawm inducer, qhov kub thiab txias induction thiab lub sijhawm induction.Yog li ntawd, feem ntau, ua ntej ib qho tsis paub cov protein tau qhia thiab ua kom huv, nws yog qhov zoo tshaj plaws los kawm lub sij hawm induction, qhov kub thiab txias thiab IPTG concentration thiaj li xaiv qhov tsim nyog thiab tau txais qhov kev sim zoo tshaj plaws.