IPTG (isopropyl-β-D-thiogalactoside) ndi chinthu chofanana ndi β-galactosidase substrate, chomwe chimathandiza kwambiri. Pansi pa kulowetsedwa kwa IPTG, choyambitsa chimatha kupanga complex ndi protein yoletsa, kotero kuti mawonekedwe a protein yoletsa asinthidwe, kotero kuti sangaphatikizidwe ndi gene yolunjika, ndipo gene yolunjika iwonetsedwe bwino. Ndiye kodi kuchuluka kwa IPTG kuyenera kudziwika bwanji panthawi yoyesera? Kodi kukula kwake ndikobwino?
Choyamba, tiyeni timvetse mfundo ya IPTG induction: Lactose operon (element) ya E. coli ili ndi majini atatu omangira, Z, Y, ndi A, omwe amalemba β-galactosidase, permease, ndi acetyltransferase, motsatana. LacZ imathira lactose mu glucose ndi galactose, kapena mu allo-lactose; lacY imalola lactose m'chilengedwe kudutsa mu nembanemba ya selo ndikulowa mu selo; lacA imasamutsa gulu la acetyl kuchokera ku acetyl-CoA kupita ku β-galactoside, zomwe zimaphatikizapo kuchotsa Poison effect. Kuphatikiza apo, pali operating sequence O, starting sequence P ndi regulatory gene I. I gene code ndi repressor protein yomwe imatha kumangirira ku malo a O a operator sequence, kotero kuti operon (meta) ichotsedwe ndikuzimitsidwa. Palinso malo omangira a catabolic gene activator protein-CAP binding site pamwamba pa initiative sequence P. P sequence, O sequence ndi CAP binding site pamodzi zimapanga regulatory region ya lac operon. Majini olembera ma enzyme atatuwa amayendetsedwa ndi dera lomwelo lolamulira kuti akwaniritse kufotokozedwa kogwirizana kwa zinthu za majini.
Ngati lactose ilibe, lac operon (meta) imakhala mu mkhalidwe woponderezedwa. Panthawiyi, lac repressor yomwe imawonetsedwa ndi I sequence motsogozedwa ndi PI promoter sequence imalumikizana ndi O sequence, yomwe imaletsa RNA polymerase kuti isagwirizane ndi P sequence ndikuletsa kuyambitsa transcription; pamene lactose ilipo, lac operon (meta) imatha kuyambitsidwa Mu operon (meta) system iyi, inducer yeniyeni si lactose yokha. Lactose imalowa mu cell ndipo imathandizidwa ndi β-galactosidase kuti isinthidwe kukhala allolactose. Yomalizayi, monga inducer molecule, imalumikizana ndi repressor protein ndikusintha kapangidwe ka protein, zomwe zimapangitsa kuti repressor protein isiyane ndi O sequence ndi transcription. Isopropylthiogalactoside (IPTG) ili ndi zotsatira zofanana ndi allolactose. Ndi inducer yamphamvu kwambiri, yomwe simagayidwa ndi mabakiteriya ndipo ndi yokhazikika kwambiri, kotero imagwiritsidwa ntchito kwambiri m'ma laboratories.
Kodi mungadziwe bwanji kuchuluka kwa IPTG komwe kuli koyenera? Tengani chitsanzo cha E. coli.
Mtundu wa E. coli BL21 wopangidwa ndi majini wokhala ndi pGEX (CGRP/msCT) wopangidwa ndi majini unayikidwa mu LB liquid medium yokhala ndi 50μg·mL-1 Amp, ndipo unalimidwa usiku wonse pa kutentha kwa 37°C. Mtundu womwe uli pamwambapa unayikidwa mu mabotolo 10 a 50mL fresh LB liquid medium yokhala ndi 50μg·mL-1 Amp pa chiŵerengero cha 1:100 kuti ukhale ndi kukula, ndipo pamene mtengo wa OD600 unali 0.6~0.8, IPTG inawonjezedwa ku kuchuluka komaliza. Ndi 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mmol·L-1. Pambuyo poyambitsa kutentha komweko komanso nthawi yomweyo, 1 mL ya yankho la bakiteriya idatengedwa kuchokera pamenepo, ndipo maselo a bakiteriya adasonkhanitsidwa pogwiritsa ntchito centrifugation ndikuyikidwa mu SDS-PAGE kuti awone momwe kuchuluka kwa IPTG kumakhudzira kuchuluka kwa mapuloteni, kenako kusankha kuchuluka kwa IPTG komwe kumakhudza kuchuluka kwa mapuloteni ambiri.
Pambuyo poyesa, zidzapezeka kuti kuchuluka kwa IPTG si kwakukulu momwe zingathere. Izi zili choncho chifukwa IPTG ili ndi poizoni winawake kwa mabakiteriya. Kupitirira kuchuluka kumeneku kudzaphanso selo; ndipo nthawi zambiri, tikukhulupirira kuti mapuloteni osungunuka kwambiri omwe amawonekera mu selo, amakhala abwino, koma nthawi zambiri pamene kuchuluka kwa IPTG kuli kwakukulu kwambiri, kuchuluka kwakukulu kwa kuphatikizidwa kudzapangidwa. Thupi, koma kuchuluka kwa mapuloteni osungunuka kumachepa. Chifukwa chake, kuchuluka kwa IPTG koyenera kwambiri nthawi zambiri sikukhala kwakukulu komwe kumakhala bwino, koma kuchepa kwa ndende.
Cholinga cha kuyambitsa ndi kukulitsa mitundu yosinthidwa majini ndikuwonjezera phindu la mapuloteni omwe akufuna ndikuchepetsa ndalama. Kuwonekera kwa jini yomwe akufuna sikungokhudzidwa ndi zinthu zomwe zimayambitsa matendawa komanso momwe plasmid imagwirira ntchito, komanso ndi zinthu zina zakunja, monga kuchuluka kwa chinthu choyambitsa, kutentha kwa chinthu choyambitsa komanso nthawi yoyambitsa. Chifukwa chake, nthawi zambiri, puloteni yosadziwika isanatulutsidwe ndikuyeretsedwa, ndibwino kuphunzira nthawi yoyambitsa, kutentha ndi kuchuluka kwa IPTG kuti musankhe mikhalidwe yoyenera ndikupeza zotsatira zabwino kwambiri zoyesera.
Nthawi yotumizira: Disembala 31-2021