IPTG (isopropyl-β-D-thiogalactoside) ni ikintu kigereranywa na β-galactosidase substrate, gishobora gukururwa cyane. Mu gihe cyo gushyiramo IPTG, inducer ishobora gukora complex hamwe na poroteyine igabanya ubukana, ku buryo imiterere ya poroteyine igabanya ubukana ihinduka, ku buryo idashobora guhuzwa na gene target, kandi gene target igaragazwa neza. None se ni gute ingano ya IPTG yagenwa mu gihe cy'igerageza? Ese nini niyo nziza kurushaho?
Ubwa mbere, reka dusobanukirwe ihame rya IPTG induction: Lactose operon (ikintu) cya E. coli kigizwe na genes eshatu z’imiterere, Z, Y, na A, zishyiraho β-galactosidase, permease, na acetyltransferase, uko bikurikirana. lacZ igabanya lactose muri glucose na galactose, cyangwa muri allo-lactose; lacY yemerera lactose mu bidukikije kunyura mu ruhu rw’uturemangingo ikajya mu turemangingo; lacA yimura itsinda rya acetyl kuva kuri acetyl-CoA ikajya kuri β-galactoside, ibyo bikaba birimo gukuraho ingaruka mbi. Byongeye kandi, hari urukurikirane rw’imikorere O, urukurikirane rw’ibanze P na genes regulatory I. Kode ya genes I ni poroteyine igabanya ubukana ishobora kwihambira ku mwanya O w’urukurikirane rw’imikorere, bityo operon (meta) igahagarikwa ikazimwa. Hariho kandi ahantu ho kwihambira hagenewe catabolic gene activator protein-CAP binding site hejuru y’urukurikirane rw’ibanze P. Urukurikirane rwa P, urukurikirane rwa O n’urukurikirane rw’uruziga rwa CAP hamwe bigize agace gashinzwe kugenzura ka lac operon. Ingirabuzimafatizo z’uturemangingo dutatu zigenzurwa n’akarere kamwe gashinzwe kugenzura kugira ngo habeho uburyo bwo guhuza ibikorwa by’uturemangingo.
Iyo lactose idafite, lac operon (meta) iba iri mu mimerere yo kugabanya. Muri iki gihe, lac repressor igaragazwa n'urukurikirane rwa I igenzurwa n'urukurikirane rwa PI promoter ruhuza n'urukurikirane rwa O, rubuza RNA polymerase gufatana n'urukurikirane rwa P kandi rukabuza gutangira kwa transcription; iyo lactose ihari, lac operon (meta) ishobora guterwa Muri iyi sisitemu ya operon (meta), inducer nyayo si lactose ubwayo. Lactose yinjira mu ngirabuzimafatizo igaterwa imbaraga na β-galactosidase kugira ngo ihindurwe allolactose. Iya nyuma, nk'umusemburo utera, ifatana na poroteyine itera kandi igahindura imiterere ya poroteyine, bigatuma poroteyine itera imbaraga itandukana n'urukurikirane rwa O na transcription. Isopropylthiogalactoside (IPTG) ifite ingaruka zimwe na allolactose. Ni inducer ikomeye cyane, idakoreshwa na bagiteri kandi irahamye cyane, bityo ikoreshwa cyane muri laboratwari.
Ni gute wamenya urugero rwiza rwa IPTG? Reka dufate urugero rwa E. coli.
Ubwoko bwa E. coli BL21 bwahinduwe mu buryo bw'uturemangingo burimo pGEX nziza (CGRP / msCT) bwashyizwe mu buryo bw'amazi bwa LB burimo 50μg·mL-1 Amp, maze buterwa mu ijoro ryose kuri 37°C. Ubu bwoko bwavuzwe haruguru bwashyizwe mu macupa 10 y'ubu bwoko bw'amazi bwa LB 50mL bushya burimo 50μg·mL-1 Amp ku gipimo cya 1:100 kugira ngo buvumburwe, kandi igihe agaciro ka OD600 kari 0.6 ~ 0.8, IPTG yongewe ku gipimo cya nyuma. 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 gukururwa ku bushyuhe bumwe no mu gihe kimwe, hafashwemo mL 1 y'umuti wa bagiteri, maze uturemangingo twa bagiteri dukusanywa hakoreshejwe centrifugation hanyuma dushyirwa kuri SDS-PAGE kugira ngo basesengure ingaruka z'ingano zitandukanye za IPTG ku mikorere ya poroteyine, hanyuma bahitamo ingano ya IPTG ifite poroteyine nyinshi.
Nyuma y'igerageza, bizagaragara ko ingano ya IPTG idahagije. Ibi biterwa nuko IPTG ifite uburozi runaka kuri bagiteri. Kurenga ingano bizica ingirabuzimafatizo; kandi muri rusange, twizeye ko poroteyine ishongesha cyane igaragara mu ngirabuzimafatizo, ari byo byiza, ariko akenshi iyo ingano ya IPTG iri hejuru cyane, ingano nini y'ingirabuzimafatizo izakorwa. Umubiri, ariko ingano ya poroteyine ishongesha yaragabanutse. Kubwibyo, ingano ya IPTG ikwiye cyane akenshi ntabwo ari nini cyane, ahubwo ni uko ingano ntoya.
Intego yo gutera no guteza imbere ubwoko bwa genetiki bwahinduwe ni ukongera umusaruro wa poroteyine igamije no kugabanya ikiguzi. Igaragara rya gene y’ingenzi ntirigira ingaruka gusa ku bintu by’ubwoko bwa gene ubwayo n’igaragara rya plasmide, ahubwo rinaterwa n’ibindi bintu byo hanze, nko kuba ingano y’ikintu kiyitera, ubushyuhe bw’ikintu n’igihe cyo kuyitera. Bityo rero, muri rusange, mbere yuko poroteyine itazwi igaragara kandi igatunganywa, ni byiza kwiga igihe cyo kuyitera, ubushyuhe n’ingano ya IPTG kugira ngo hamenyekane imiterere ikwiye kandi haboneke ibisubizo byiza by’igerageza.
Igihe cyo kohereza: Ukuboza 31-2021