labarai

IPTG (isopropyl-β-D-thiogalactoside) analog ne na β-galactosidase substrate, wanda ba shi da ƙarfi sosai.Ƙarƙashin ƙaddamar da IPTG, mai ƙaddamarwa zai iya samar da hadaddun tare da furotin mai raɗaɗi , Don haka an canza nau'in furotin mai mahimmanci, don haka ba za a iya haɗa shi tare da kwayar da aka yi niyya ba, kuma an bayyana maƙasudin manufa da kyau.Don haka ta yaya ya kamata a ƙayyade ƙaddamarwar IPTG yayin gwajin?Shin mafi girma shine mafi kyau?
Da farko, bari mu fahimci ka'idar shigar da IPTG: E. coli's lactose operon (element) yana ƙunshe da kwayoyin halitta guda uku, Z, Y, da A, waɗanda ke ɓoye β-galactosidase, permease, da acetyltransferase, bi da bi.lacZ hydrolyzes lactose cikin glucose da galactose, ko cikin allo-lactose;lacY yana ba da damar lactose a cikin yanayi don wucewa ta cikin kwayar halitta kuma ya shiga cikin tantanin halitta;lacA yana canza ƙungiyar acetyl daga acetyl-CoA zuwa β-galactoside, wanda ya haɗa da cire sakamako mai guba.Bugu da ƙari, akwai tsarin aiki O, jerin farawa P da tsarin tsara tsarin I. I gene code shine furotin mai dannewa wanda zai iya ɗaure zuwa matsayi O na jerin ma'aikata, don haka an danne operon (meta) kashe.Har ila yau, akwai wurin ɗaure don mahaɗar furotin-CAP mai ɗauri na catabolic gene activator a sama na tsarin farawa P. Jerin P, jerin O da CAP daurin wurin tare sun zama yankin da aka tsara na lac operon.Kwayoyin codeing na enzymes guda uku ana sarrafa su ta hanyar yanki guda ɗaya don cimma daidaituwar bayanin samfuran kwayoyin halitta.

Idan babu lactose, lac operon (meta) yana cikin yanayin danniya.A wannan lokacin, lac repressor da aka bayyana ta hanyar I a ƙarƙashin kulawar tsarin mai gabatarwa na PI yana ɗaure zuwa jerin O, wanda ke hana RNA polymerase ɗaure zuwa jerin P kuma yana hana farawa rubutu;Lokacin da lactose ya kasance, ana iya haifar da lac operon (meta) A cikin wannan tsarin operon (meta), ainihin inducer ba lactose kanta ba ne.Lactose yana shiga cikin tantanin halitta kuma an daidaita shi ta β-galactosidase don canza shi zuwa allolactose.Na ƙarshe, a matsayin ƙwayar inducer, yana ɗaure da furotin mai matsawa kuma yana canza tsarin furotin, wanda ke haifar da rarrabuwar furotin mai ƙarfi daga jerin O da rubutun.isopropylthiogalactoside (IPTG) yana da tasiri iri ɗaya kamar allolactose.Yana da matukar ƙarfi inducer, wanda ba a daidaita shi ta hanyar ƙwayoyin cuta kuma yana da ƙarfi sosai, don haka ana amfani dashi sosai a cikin dakunan gwaje-gwaje.

Yadda za a ƙayyade mafi kyau duka taro na IPTG?Dauki E. coli a matsayin misali.
E. coli BL21 nau'in injiniyar kwayoyin halitta mai dauke da ingantaccen pGEX (CGRP/msCT) an saka shi cikin ruwa na LB mai dauke da 50μg · mL-1 Amp, kuma an yi al'ada a cikin dare a 37 ° C.An shigar da al'adun da ke sama a cikin kwalabe 10 na 50mL sabo LB mai matsakaicin ruwa mai dauke da 50μg · mL-1 Amp a wani rabo na 1: 100 don al'adun fadada, kuma lokacin da darajar OD600 ta kasance 0.6 ~ 0.8, an ƙara IPTG zuwa taro na ƙarshe.Yana da 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mmol·L-1.Bayan shigar da shi a cikin zafin jiki guda ɗaya da lokaci guda, an ɗauki 1 ml na maganin ƙwayar cuta daga gare ta, kuma an tattara ƙwayoyin ƙwayoyin cuta ta hanyar centrifugation kuma an sanya su zuwa SDS-PAGE don nazarin tasirin tasirin IPTG daban-daban akan maganganun furotin, sannan zaɓi ƙaddamarwar IPTG tare da mafi girman bayanin furotin.
Bayan gwaje-gwaje, za a gano cewa ƙaddamarwar IPTG ba ta da girma kamar yadda zai yiwu.Wannan saboda IPTG yana da takamaiman guba ga ƙwayoyin cuta.Yin wuce gona da iri kuma zai kashe tantanin halitta;kuma gabaɗaya magana, muna fatan cewa furotin mai narkewa da aka bayyana a cikin tantanin halitta, mafi kyau, amma a yawancin lokuta lokacin da maida hankali na IPTG ya yi yawa, za a sami babban adadin haɗawa.Jiki, amma adadin furotin mai narkewa ya ragu.Sabili da haka, mafi dacewa ƙaddamarwar IPTG sau da yawa ba shine mafi girma ba, amma ƙananan ƙaddamarwa.
Manufar ƙaddamarwa da noma nau'ikan nau'ikan injiniyoyin halitta shine haɓaka yawan amfanin furotin da aka yi niyya da rage farashi.Bayanin da dalihan da maganganun da ke da shi ba ya shafa kawai da kuma furucin da wasu kuma wasu sharadin waje, kamar su maida hankali da rashin daidaituwa da lokacin shiga.Sabili da haka, gabaɗaya, kafin an bayyana furotin da ba a sani ba kuma an tsarkake shi, yana da kyau a yi nazarin lokacin ƙaddamarwa, zafin jiki da kuma maida hankali na IPTG don zaɓar yanayin da ya dace da kuma samun sakamako mafi kyau na gwaji.