amakuru

IPTG (isopropyl-β-D-thiogalactoside) ni igereranya rya β-galactosidase substrate, idashoboka cyane.Mugihe cyo kwinjiza IPTG, inducer irashobora gukora urwego rugizwe na poroteyine ya repressor, Ku buryo ihinduka rya poroteyine ya repressor ihinduka, ku buryo ridashobora guhuzwa na gen igenewe, kandi gene igaragazwa neza.Nigute intumbero ya IPTG igomba kugenwa mugihe cyigeragezo?Ninini nini nziza?
Ubwa mbere, reka twumve ihame ryo kwinjiza IPTG: E. coli ya lactose operon (element) ikubiyemo genes eshatu zubatswe, Z, Y, na A, zikubiyemo β-galactosidase, permease, na acetyltransferase.lacZ hydrolyzes lactose muri glucose na galaktose, cyangwa muri allo-lactose;lacY yemerera lactose mubidukikije kunyura muri selile hanyuma ikinjira muri selire;lacA yimura itsinda rya acetyl kuva acetyl-CoA kuri β-galactoside, ikubiyemo gukuraho ingaruka zuburozi.Mubyongeyeho, hariho urutonde rukora O, urutonde rwintangiriro P na gen igenga I. I code ya I ni proteine ​​ya repressor ishobora guhuza umwanya O wurwego rwabakurikirana, kugirango operon (meta) ihagarikwa kandi yazimye.Hariho kandi urubuga ruhuza catabolike ya gene ikora poroteyine-CAP ihuza urubuga hejuru yurwego rwo gutangiza P. Urutonde rwa P, O urukurikirane hamwe na CAP ihuza urubuga hamwe bigize akarere kagenzura lac operon.Imikorere ya coding ya enzymes eshatu igengwa nakarere kamwe kayobora kugirango igere ku guhuza ibicuruzwa bya gene.

Mugihe habuze lactose, lac operon (meta) iri muburyo bwo gukandamizwa.Muri iki gihe, repressor ya lac yagaragajwe nuruhererekane rwa I iyobowe na PI promoter ikurikirana ihuza na O ikurikiranye, ikabuza polymerase ya RNA guhuza na P ikabuza gutangiza inyandiko;iyo lactose ihari, lac operon (meta) irashobora guterwa Muri iyi sisitemu ya operon (meta), inducer nyayo ntabwo ari lactose ubwayo.Lactose yinjira muri selire kandi itangizwa na β-galactosidase kugirango ihindurwe kuri allolactose.Iheruka, nka molekile ya inducer, ihuza poroteyine ya repressor kandi ihindura poroteyine ihinduka, biganisha ku gutandukana kwa poroteyine ya repressor kuva kuri O ikurikirana no kwandukura.Isopropylthiogalactoside (IPTG) igira ingaruka zimwe na allolactose.Ni inducer ikomeye cyane, idakoreshwa na bagiteri kandi ihagaze neza, kuburyo ikoreshwa cyane muri laboratoire.

Nigute ushobora kumenya uburyo bwiza bwa IPTG?Fata E. coli nk'urugero.
E. coli BL21 yakozwe mubwoko bwa genetike irimo pomeX nziza (CGRP / msCT) yatewe mumashanyarazi ya LB irimo 50μg · mL-1 Amp, kandi yaraye ijoro ryose kuri 37 ° C.Umuco wavuzwe haruguru washyizwe mumacupa 10 ya 50mL mashya ya LB yamazi arimo 50μg · mL-1 Amp ku kigereranyo cya 1: 100 kumuco wo kwaguka, kandi mugihe agaciro ka OD600 kari 0,6 ~ 0.8, IPTG yongewe kumurongo wanyuma.Ni 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mmol·L-1.Nyuma yo kwinjizwa mubushuhe bumwe kandi mugihe kimwe, mL 1 yumuti wa bagiteri yakuweho, hanyuma bagiteri yakusanyirijwe hamwe na centrifugation hanyuma ikorerwa SDS-PAGE kugirango isesengure ingaruka ziterwa na IPTG zitandukanye mumagambo ya poroteyine, hanyuma hitamo IPTG yibanze hamwe na protein nini nini.
Nyuma yubushakashatsi, bizagaragara ko kwibumbira hamwe kwa IPTG atari binini bishoboka.Ni ukubera ko IPTG ifite uburozi runaka kuri bagiteri.Kurenza kwibanda nabyo bizica selile;kandi muri rusange tuvuze, twizera ko proteine ​​nyinshi zishonga zigaragara muri selile, nibyiza, ariko mubihe byinshi iyo kwibumbira hamwe kwa IPTG ari byinshi cyane, hazashyirwaho umubare munini wo kubishyiramo.Umubiri, ariko ubwinshi bwa poroteyine zishonga bwaragabanutse.Kubwibyo, kwibanda cyane kuri IPTG ntabwo arinini nini nziza, ariko hasi yibitekerezo.
Intego yo kwinjiza no guhinga ubwoko bwa geneti yakozwe na genetique ni ukongera umusaruro wa poroteyine yagenewe no kugabanya ibiciro.Imvugo ya gen igamije ntabwo ihindurwa gusa nimpamvu ziterwa nubwoko bwa plasmid, ahubwo binaterwa nizindi miterere yo hanze, nko kwibanda kwa inducer, ubushyuhe bwinjira nigihe cyo kwinjira.Kubwibyo, muri rusange, mbere yuko poroteyine itazwi igaragazwa kandi igahumanurwa, nibyiza kwiga igihe cyo kwinjiza, ubushyuhe hamwe nubushakashatsi bwa IPTG kugirango uhitemo ibihe bikwiye kandi ubone ibisubizo byiza byubushakashatsi.