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IPTG (isopropyl-β-D-thiogalactoside) inonzi analogue ye β-galactosidase substrate, iyo inonyanya kushandiswa.Pasi pekutsvaga kweIPTG, inducer inogona kuumba yakaoma neprotein repressor , Saka kuti kuumbwa kweprotein repressor kuchinjwa, kuitira kuti irege kusanganiswa nejini rinotarirwa, uye jini rinotaridzirwa rinoratidzwa zvakanaka.Saka iko kusungirirwa kweIPTG kunofanirwa kutariswa sei panguva yekuyedza?Kukura kuri nani here?
Chekutanga, ngatinzwisisei musimboti weIPTG induction: E. coli's lactose operon (element) ine matatu emajini emuumbirwo, Z,Y, uye A, ayo encode β-galactosidase, permease, uye acetyltransferase, zvichiteerana.lacZ inoisa lactose muglucose uye galactose, kana muallo-lactose;lacY inobvumira lactose munharaunda kuti ipfuure nepakati pesero membrane uye inopinda muchitokisi;lacA inotamisa boka reacetyl kubva kuacetyl-CoA kuenda ku β-galactoside, iyo inosanganisira kubvisa Toxic effect.Mukuwedzera, kune kutevedzana kwekushanda O, kutevedzana kwekutanga P uye gene regulatory I. I gene gene code iprotein repressor inogona kusunga panzvimbo yeO yekutevedzana kwevashandi, kuitira kuti operon (meta) adzvinyirwe uye yakadzimwa.Panewo saiti inosunga ye catabolic gene activator protein-CAP binding site kumusoro kwekutanga kutevedzana P. The P sequence, O sequence uye CAP inosunga nzvimbo pamwe chete inoumba dunhu rekutonga re lac operon.Iwo macoding majini ema enzymes matatu anodzorwa neiyo dunhu rekutonga kuti riwane kurongeka kwekutaura kwemajini zvigadzirwa.

Kana pasina lactose, lac operon (meta) iri mumamiriro ekudzvinyirira.Panguva ino, iyo lac repressor inoratidzwa neI sequence pasi pekutonga kwePI kukurudzira kutevedzana kunosunga kuO sequence, iyo inodzivirira RNA polymerase kubva pakusunga kuP sequence uye inhibits kutangisa kunyorwa;kana lactose iripo, iyo lac operon (meta) inogona kukonzerwa Mune iyi operon (meta) system, inducer chaiyo haisi lactose pachayo.Lactose inopinda muchitokisi uye inogadziriswa ne β-galactosidase kuti ishandurwe kuita allolactose.Iyo yekupedzisira, seye inducer molecule, inosungira kune repressor protein uye inoshandura mapuroteni conformation, izvo zvinotungamirira mukuparadzaniswa kweprotein repressor kubva kuO sequence uye chinyorwa.Isopropylthiogalactoside (IPTG) ine mhedzisiro yakafanana nealllolactose.Iyo inducer ine simba kwazvo, iyo isina metabolized nebhakitiriya uye yakasimba kwazvo, saka inoshandiswa zvakanyanya mumarabhoritari.

Nzira yekuziva sei iyo yakakwana yekusangana kweIPTG?Tora E. coli semuenzaniso.
The E. coli BL21 genetically engineered strain ine positive recombinant pGEX (CGRP/msCT) yakaiswa muLB liquid medium ine 50μg · mL-1 Amp, uye yakagadzirwa usiku hwose pa37 ° C.Tsika dziri pamusoro dzakaiswa mumabhodhoro e10 e50mL matsva eLB liquid medium ine 50μg · mL-1 Amp pachiyero che1: 100 yekuwedzera tsika, uye apo OD600 inokosha yaiva 0.6 ~ 0.8, IPTG yakawedzerwa kune yekupedzisira.Zviri 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mmol · L-1.Mushure mekuiswa pakushisa kwakafanana uye panguva imwechete, 1 mL yehutachiona hwehutachiona yakatorwa kubva mairi, uye masero ebhakitiriya akaunganidzwa ne centrifugation uye akaiswa pasi peSDS-PAGE kuti aongorore simba rezvikamu zvakasiyana zve IPTG pamapuroteni kutaura, uye ipapo. sarudza iyo IPTG yekumisikidza ine yakakura mapuroteni kutaura.
Mushure mekuedza, zvichazoonekwa kuti kusungirirwa kweIPTG hakuna kukura sezvinobvira.Izvi zvinodaro nekuti IPTG ine imwe huturu kune mabhakitiriya.Kupfuura kusungirirwa kuchaurayawo sero;uye kazhinji kutaura, tinotarisira kuti puroteni yakanyunguduka inoratidzwa musero, zviri nani, asi muzviitiko zvakawanda apo kuiswa kweIPTG kwakanyanyisa, huwandu hwakawanda hwekubatanidzwa huchaumbwa.Muviri, asi huwandu hweprotein inonyungudika hwakadzikira.Naizvozvo, iyo yakanyanya kufanirwa IPTG yekumisikidza kazhinji haisi iyo yakakura zvirinani, asi yakaderera iyo yekumisikidza.
Chinangwa chekupinza uye kurima genetically engineered strains ndechekuwedzera goho reprotein inotarirwa uye kuderedza mitengo.Chirevo chejini chinotarirwa hachingogadziriswi chete neiyo strain's pachayo zvinhu uye kutaura plasmid, asiwo nemamwe mamiriro ekunze, akadai sekusangana kweinducer, tembiricha yekupinza uye induction nguva.Nokudaro, kazhinji, musati puroteni isingazivikanwe isati yaratidzwa uye yakacheneswa, zviri nani kudzidza nguva yekupinza, tembiricha uye IPTG yekumisikidza kuitira kuti usarudze mamiriro akakodzera uye uwane mhinduro dzakanakisa dzekuyedza.