IPTG (isopropyl-β-D-thiogalactoside) kwatancen substrate ne na β-galactosidase, wanda yake da matuƙar tasiri. A ƙarƙashin shigar IPTG, mai haifar da shi zai iya samar da wani hadadden tsari tare da furotin mai rage zafi, Ta yadda za a canza yanayin furotin mai rage zafi, ta yadda ba za a iya haɗa shi da kwayar halittar da ake nufi ba, kuma a bayyana kwayar halittar da ake nufi yadda ya kamata. To ta yaya ya kamata a tantance yawan IPTG a lokacin gwajin? Shin girman ya fi kyau?
Da farko, bari mu fahimci ƙa'idar shigar da IPTG: lactose operon (element) na E. coli ya ƙunshi kwayoyin halitta guda uku na tsari, Z, Y, da A, waɗanda ke ƙunshe da β-galactosidase, permease, da acetyltransferase, bi da bi. lacZ yana ƙara lactose zuwa glucose da galactose, ko kuma zuwa allo-lactose; lacY yana ba da damar lactose a cikin muhalli ya ratsa ta cikin membrane na tantanin halitta kuma ya shiga tantanin halitta; lacA yana canja wurin ƙungiyar acetyl daga acetyl-CoA zuwa β-galactoside, wanda ya haɗa da cire tasirin Toxic. Bugu da ƙari, akwai jerin aiki O, jerin farawa P da kuma kwayar halitta mai tsari I. Lambar kwayar halitta ta I furotin ne mai hana ruwa wanda zai iya ɗaurewa zuwa matsayin O na jerin aiki, don haka ana danne operon (meta) kuma a kashe shi. Akwai kuma wurin ɗaurewa don wurin ɗaure furotin-CAP mai kunna kwayar halitta daga sama da jerin farawa na P. Jerin P, jerin O da wurin ɗaure CAP tare sun ƙunshi yankin da ke kula da lac operon. Ana tsara kwayoyin halittar code na enzymes guda uku ta hanyar yankin da ke kula da kwayoyin halitta iri ɗaya don cimma daidaiton bayyanar samfuran kwayoyin halitta.
Idan babu lactose, lac operon (meta) yana cikin mawuyacin hali. A wannan lokacin, lac repressor da aka bayyana ta hanyar jerin I a ƙarƙashin ikon jerin masu haɓaka PI yana ɗaure zuwa jerin O, wanda ke hana RNA polymerase ɗaure zuwa jerin P kuma yana hana fara rubutu; lokacin da lactose yake, ana iya haifar da lac operon (meta) A cikin wannan tsarin operon (meta), ainihin mai haifar da ba lactose bane. Lactose yana shiga tantanin halitta kuma β-galactosidase yana haɓaka shi don a canza shi zuwa allolactose. Na ƙarshen, a matsayin ƙwayar mai haifar da shi, yana ɗaure da furotin mai matsewa kuma yana canza yanayin furotin, wanda ke haifar da rabuwar furotin mai matsewa daga jerin O da kwafi. Isopropylthiogalactoside (IPTG) yana da tasiri iri ɗaya da allolactose. Yana da mai haifar da ƙarfi sosai, wanda ba a metabolize shi ta hanyar ƙwayoyin cuta kuma yana da ƙarfi sosai, don haka ana amfani da shi sosai a dakunan gwaje-gwaje.
Yaya za a tantance mafi kyawun yawan IPTG? Ka ɗauki E. coli a matsayin misali.
An yi wa nau'in E. coli BL21 da aka ƙera ta hanyar halitta wanda ke ɗauke da pGEX mai kyau (CGRP/msCT) allura a cikin ruwan LB mai ɗauke da 50μg·mL-1 Amp, sannan aka dasa shi cikin dare a zafin jiki na 37°C. An yi wa wannan nau'in allura a cikin kwalaben ruwa 10 na ruwan LB mai sabo mai ɗauke da 50μg·mL-1 Amp a rabo na 1:100 don faɗaɗawa, kuma lokacin da ƙimar OD600 ta kasance 0.6~0.8, an ƙara IPTG zuwa ga yawan da aka samu. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mmol·L-1. Bayan an shigar da shi a yanayin zafi iri ɗaya da kuma lokaci guda, an ɗauki 1 mL na maganin ƙwayoyin cuta daga gare shi, kuma an tattara ƙwayoyin ƙwayoyin cuta ta hanyar centrifugation kuma an yi musu allurar SDS-PAGE don yin nazarin tasirin yawan IPTG daban-daban akan bayyanar furotin, sannan a zaɓi yawan IPTG tare da mafi girman bayyanar furotin.
Bayan gwaje-gwaje, za a gano cewa yawan IPTG bai kai girman da zai yiwu ba. Wannan saboda IPTG yana da wani guba ga ƙwayoyin cuta. Fiye da yawan zai kuma kashe ƙwayar; kuma gabaɗaya, muna fatan cewa furotin mai narkewa da aka bayyana a cikin ƙwayar, zai fi kyau, amma a lokuta da yawa lokacin da yawan IPTG ya yi yawa, za a samar da adadi mai yawa na haɗawa. Jiki, amma adadin furotin mai narkewa ya ragu. Saboda haka, yawan IPTG da ya fi dacewa sau da yawa ba shine mafi girma ba, mafi kyau, amma ƙasa da yawan haɗuwa.
Manufar shigar da kuma haƙa nau'ikan da aka ƙera ta hanyar halitta ita ce ƙara yawan furotin da ake buƙata da kuma rage farashi. Bayyanar kwayar halittar da ake buƙata ba wai kawai tana shafar abubuwan da ke haifar da ita da kuma bayyanar plasmid ba, har ma da wasu yanayi na waje, kamar yawan sinadarin da ke haifar da ita, zafin jiki da lokacin shigar da ita. Saboda haka, gabaɗaya, kafin a bayyana kuma a tsarkake wani furotin da ba a sani ba, ya fi kyau a yi nazarin lokacin shigar da ita, zafin jiki da kuma yawan sinadarin IPTG domin zaɓar yanayin da ya dace da kuma samun mafi kyawun sakamakon gwaji.
Lokacin Saƙo: Disamba-31-2021