litaba

IPTG (isopropyl-β-D-thiogalactoside) ke analogue ea substrate ea β-galactosidase, e kentsoeng haholo.Tlas'a ho kenngoa ha IPTG, inducer e ka theha e rarahaneng le protheine ea khatello , E le hore ho fetoloa ha protheine ea khatello ea maikutlo ho fetoloe, e le hore e se ke ea kopanngoa le liphatsa tsa lefutso, 'me liphatsa tsa lefutso tse lebisitsoeng li hlahisoa ka mokhoa o nepahetseng.Joale mahloriso a IPTG a lokela ho khethoa joang nakong ea teko?Na e kholoanyane e molemo?
Taba ea pele, ha re utloisise molao-motheo oa IPTG induction: E. coli's lactose operon (element) e na le mefuta e meraro ea liphatsa tsa lefutso, Z,Y, le A, tse kenyelletsang β-galactosidase, permease, le acetyltransferase, ka ho latellana.lacZ hydrolyze lactose hore e be tsoekere le galactose, kapa ho allo-lactose;lacY e lumella lactose tikolohong hore e fete ka har'a lera la sele ebe e kena seleng;lacA e fetisetsa sehlopha sa acetyl ho tloha ho acetyl-CoA ho ea β-galactoside, e kenyelletsang ho tlosa phello ea Chefo.Ho phaella moo, ho na le tatellano ea ts'ebetso ea O, tatellano ea ho qala P le liphatsa tsa lefutso tsa I. I gene code ke protheine e hatellang e ka tlamang boemo ba O ea tatellano ea opareitara, e le hore operon (meta) e hatellehe le ho hatelloa. tima.Ho boetse ho na le sebaka se tlamang bakeng sa sebaka se tlamang sa catabolic gene activator protein-CAP e holimo ho tatellano e qalang P. Sebaka sa P. Tatelano ea O le sebaka se tlamang sa CAP hammoho li etsa sebaka sa taolo ea lac operon.Liphatsa tsa lefutso tsa likhoutu tsa li-enzyme tse tharo li laoloa ke sebaka se le seng sa taolo ho fihlela polelo e hokahaneng ea lihlahisoa tsa lefutso.

Ha lactose e le sieo, lac operon (meta) e boemong ba khatello.Ka nako ena, mohatelli oa lac o hlalositsoeng ke tatellano ea I tlas'a taolo ea tatellano ea PI e khothalletsang ho latellana ha O, e thibelang RNA polymerase hore e se ke ea tlama ho P le ho thibela ho qalisoa ha transcript;ha lactose e le teng, lac operon (meta) e ka etsoa Tsamaisong ena ea operon (meta), inducer ea sebele hase lactose ka boeona.Lactose e kena ka seleng 'me e hlahisoa ke β-galactosidase hore e fetoleloe ho allolactose.E qetellang, e le molek'hule ea inducer, e tlama ho protheine ea khatello 'me e fetola protheine conformation, e lebisang ho arohana ha protheine ea khatello ho tloha ho O tatellano le ho ngoloa.Isopropylthiogalactoside (IPTG) e na le phello e tšoanang le ea allolactose.Ke inducer e matla haholo, e sa sebelisoang ke libaktheria 'me e tsitsitse haholo, kahoo e sebelisoa haholo li laboratories.

U ka tseba joang hore na IPTG e nepahetse hakae?Nka E. coli e le mohlala.
E. coli BL21 e entsoeng ka liphatsa tsa lefutso e nang le "positive recombinant pGEX" (CGRP/msCT) e ile ea kenngoa ka har'a metsi a LB a nang le 50μg·mL-1 Amp, 'me a hlahisoa bosiu bo le bong ka 37 ° C.Tloaelo e ka holimo e ne e kenngoa ka har'a libotlolo tsa 10 tsa 50mL e ncha ea LB liquid medium e nang le 50μg · mL-1 Amp ka karolelano ea 1: 100 bakeng sa setso sa ho atolosa, 'me ha boleng ba OD600 e ne e le 0.6 ~ 0.8, IPTG e ile ea kenngoa mohopolong oa ho qetela.Ke 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mmol · L-1.Ka mor'a ho kenngoa ka mocheso o le mong le ka nako e ts'oanang, 1 mL ea tharollo ea baktheria e nkiloe ho eona, 'me lisele tsa baktheria li ile tsa bokelloa ka centrifugation' me li tlas'a SDS-PAGE ho hlahloba tšusumetso ea likhahla tse fapaneng tsa IPTG mabapi le polelo ea protheine, ebe joale. khetha khatello ea IPTG e nang le polelo e kholo ea protheine.
Kamora liteko, ho tla fumanoa hore khatello ea IPTG ha e kholo ka moo ho ka khonehang.Sena ke hobane IPTG e na le chefo e itseng ho baktheria.Ho feta ho tsepamisa mohopolo ho tla boela ho bolaee sele;'me ka kakaretso re bua, re tšepa hore protheine e qhibilihang haholo e hlahisoang ka seleng, e molemo, empa maemong a mangata ha khatello ea IPTG e phahame haholo, palo e kholo ea kenyelletso e tla thehoa.'Mele, empa palo ea protheine e qhibilihang e fokotsehile.Ka hona, khatello e nepahetseng ka ho fetisisa ea IPTG hangata ha e kholo ho feta, empa e theola khatello ea maikutlo.
Sepheo sa ho kenya le ho lema mefuta e entsoeng ka liphatsa tsa lefutso ke ho eketsa chai ea protheine e lebisitsoeng le ho fokotsa litšenyehelo.Polelo ea liphatsa tsa lefutso tse lebisitsoeng ha e ame feela ke lintlha tsa khatello le polelo ea plasmid, empa hape le maemo a mang a kantle, joalo ka khatello ea induction, mocheso oa induction le nako ea ho kenella.Ka hona, ka kakaretso, pele protheine e sa tsejoeng e hlahisoa le ho hloekisoa, ho molemo ho ithuta nako ea ho kenngoa, mocheso le khatello ea IPTG e le hore u khethe maemo a loketseng le ho fumana liphello tse ntle ka ho fetisisa tsa liteko.