nkhani

IPTG (isopropyl-β-D-thiogalactoside) ndi analogue ya gawo lapansi la β-galactosidase, lomwe limakhala losavuta kwambiri.Pansi pa kulowetsedwa kwa IPTG, inducer ikhoza kupanga zovuta ndi mapuloteni opondereza , Kotero kuti conformation ya mapuloteni opondereza asinthe, kotero kuti sangathe kuphatikizidwa ndi jini yowunikira, ndipo jini yowunikira imasonyezedwa bwino.Ndiye kodi kuchuluka kwa IPTG kuyenera kuzindikirika bwanji panthawi yoyeserera?Kodi chachikulu ndi chabwino?
Choyamba, tiyeni timvetse mfundo ya IPTG induction: E. coli's lactose operon (elementi) ili ndi majini atatu opangidwa, Z,Y, ndi A, omwe amalowetsa β-galactosidase, permease, ndi acetyltransferase, motsatira.lacZ hydrolyzes lactose kukhala shuga ndi galactose, kapena allo-lactose;lacY imalola lactose m'chilengedwe kudutsa mu cell membrane ndikulowa mu cell;lacA imasamutsa gulu la acetyl kuchoka ku acetyl-CoA kupita ku β-galactoside, zomwe zimaphatikizapo kuchotsa Poizoni.Kuonjezera apo, pali ndondomeko yogwiritsira ntchito O, ndondomeko yoyambira P ndi jini yolamulira I. I gene code ndi mapuloteni opondereza omwe amatha kumangirira ku malo O a ndondomeko ya oyendetsa, kotero kuti operon (meta) amaponderezedwa ndipo kuzimitsa.Palinso malo omangirira a catabolic gene activator protein-CAP binding site on the intiative sequence P. The P sequence, O sequence and CAP bining site together are the regulatory region of lac operon.Ma jini olembera a ma enzyme atatuwa amayendetsedwa ndi gawo lowongolera lomwelo kuti akwaniritse mawonekedwe ogwirizana azinthu zamtundu.

Pakakhala lactose, lac operon (meta) imakhala yoponderezedwa.Panthawiyi, lac repressor yomwe imasonyezedwa ndi ndondomeko ya I pansi pa ulamuliro wa PI wotsatsira ndondomeko imamangiriza ku mndandanda wa O, zomwe zimalepheretsa RNA polymerase kuti isamangirire ku ndondomeko ya P ndikuletsa kuyambika kwa transcript;pamene lactose ilipo, lac operon (meta) ikhoza kupangitsidwa Mu dongosolo la operon (meta), choyambitsa chenichenicho si lactose yokha.Lactose imalowa m'selo ndipo imapangidwa ndi β-galactosidase kuti isinthidwe kukhala allolactose.Chotsatiracho, monga molekyu ya inducer, imamangiriza ku mapuloteni opondereza ndikusintha mapuloteni, omwe amachititsa kuti mapuloteni opondereza awonongeke kuchokera ku mndandanda wa O ndi kulembedwa.Isopropylthiogalactoside (IPTG) imakhala ndi zotsatira zofanana ndi allolactose.Ndiwothandizira kwambiri, omwe samapangidwa ndi mabakiteriya ndipo amakhala okhazikika, choncho amagwiritsidwa ntchito kwambiri m'ma laboratories.

Kodi mungadziwe bwanji kuchuluka kwa IPTG?Tengani E. coli mwachitsanzo.
Mtundu wa E. coli BL21 wopangidwa ndi chibadwa wokhala ndi recombinant yabwino pGEX (CGRP/msCT) unalowetsedwa mu LB madzi sing'anga yomwe ili ndi 50μg·mL-1 Amp, ndi kutukuka usiku wonse pa 37 ° C.Chikhalidwe chomwe chili pamwambachi chinalowetsedwa m'mabotolo a 10 a 50mL atsopano a LB amadzimadzi amadzimadzi okhala ndi 50μg · mL-1 Amp pa chiŵerengero cha 1: 100 chifukwa cha chikhalidwe chokulitsa, ndipo pamene mtengo wa OD600 unali 0.6 ~ 0.8, IPTG inawonjezeredwa ku ndende yomaliza.Ndi 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mmol · L-1.Pambuyo pa kulowetsedwa pa kutentha komweko komanso nthawi yomweyo, 1 mL ya njira ya bakiteriya inatengedwa kuchokera mmenemo, ndipo maselo a bakiteriya anasonkhanitsidwa ndi centrifugation ndi kuperekedwa kwa SDS-PAGE kuti afufuze kukhudzidwa kwa mitundu yosiyanasiyana ya IPTG pa mapuloteni, ndiyeno. sankhani kuchuluka kwa IPTG komwe kumakhala ndi protein yayikulu kwambiri.
Pambuyo pakuyesa, zidzapezeka kuti kuchuluka kwa IPTG sikuli kwakukulu momwe kungathekere.Izi zili choncho chifukwa IPTG ili ndi kawopsedwe kena ka mabakiteriya.Kupitilira ndende kudzaphanso selo;ndipo kawirikawiri, tikuyembekeza kuti mapuloteni osungunuka kwambiri omwe amawonetsedwa mu selo, amakhala bwino, koma nthawi zambiri pamene IPTG imakhala yochuluka kwambiri, kuchuluka kwa kuphatikizidwa kudzapangidwa.Thupi, koma kuchuluka kwa mapuloteni osungunuka kunachepa.Chifukwa chake, ndende yoyenera kwambiri ya IPTG nthawi zambiri sikhala yokulirapo, koma yotsika kwambiri.
Cholinga cha induction ndi kulima mitundu yopangidwa ndi majini ndi kuonjezera zokolola za mapuloteni omwe akuwunikira ndikuchepetsa mtengo.Mafotokozedwe a jini yowunikira samakhudzidwa kokha ndi zovuta zake zokha komanso mawu akuti plasmid, komanso ndi zina zakunja, monga kuchuluka kwa inducer, kutentha kwa induction ndi nthawi yolowera.Choncho, kawirikawiri, puloteni yosadziwika isanayambe kufotokozedwa ndi kuyeretsedwa, ndi bwino kuphunzira nthawi yowonetsera, kutentha ndi IPTG kuti musankhe mikhalidwe yoyenera ndikupeza zotsatira zabwino zoyesera.