IPTG (isopropyl-β-D-thiogalactoside) imhando yeβ-galactosidase substrate, iyo inonyungudika zvakanyanya. Pasi peIPTG, chinhu chinokurudzira chinogona kugadzira chinhu chakafanana neprotein inodzvinyirira, kuitira kuti chimiro cheprotein inodzvinyirira chishandurwe, kuitira kuti isakwanise kusanganiswa negene rinotarwa, uye gene rinotarwa riratidzwe zvinobudirira. Saka huwandu hweIPTG hunofanira kutsanangurwa sei panguva yekuedza? Kukura kuri nani here?
Kutanga, ngatinzwisisei musimboti weIPTG induction: Lactose operon (element) yeE. coli ine majini matatu ekuvaka, Z, Y, naA, ayo anoumba β-galactosidase, permease, uye acetyltransferase, zvichiteerana. lacZ inonyungudutsa lactose kuita glucose negalactose, kana kuti kuita allo-lactose; lacY inobvumira lactose iri munharaunda kuti ipfuure nepa membrane yesero yopinda musero; lacA inotamisa boka reacetyl kubva kuacetyl-CoA kuenda kuβ-galactoside, izvo zvinosanganisira kubvisa Toxic effect. Pamusoro pezvo, kune operating sequence O, starting sequence P uye regulatory gene I. I gene code irepressor protein inogona kusunga panzvimbo O ye operator sequence, kuitira kuti operon (meta) idzvinyirirwe uye idzimwe. Kunewo binding site ye catabolic gene activator protein-CAP binding site kumusoro kwe initiative sequence P. P sequence, O sequence uye CAP binding site pamwe chete zvinoumba regulatory region yelac operon. Majini ekunyora ma enzyme matatu anodzorwa nenzvimbo imwecheteyo yekutonga kuti awane kuratidzwa kwakarongeka kwezvigadzirwa zvemajini.
Kana pasina lactose, lac operon (meta) inenge iri mumamiriro ekudzvinyirirwa. Panguva ino, lac repressor inoratidzwa neI sequence iri pasi pekutonga kwePI promoter sequence inosunga kuO sequence, iyo inodzivirira RNA polymerase kuti isasungane neP sequence uye inodzivisa kutanga kwetranscription; kana lactose iripo, lac operon (meta) inogona kukonzerwa. Mune iyi operon (meta) system, inducer chaiyo haisi lactose pachayo. Lactose inopinda musero uye inokonzereswa neβ-galactosidase kuti ishandurwe kuita allolactose. Iyo yekupedzisira, se inducer molecule, inosunga ku repressor protein uye inoshandura chimiro cheprotein, izvo zvinotungamira mukuparadzaniswa kwerepressor protein kubva kuO sequence uye transcription. Isopropylthiogalactoside (IPTG) ine mhedzisiro yakafanana neallolactose. I inducer ine simba kwazvo, isingagayiwe nemabhakitiriya uye yakagadzikana kwazvo, saka inoshandiswa zvakanyanya mumarabhoritari.
Nzira yekuziva sei huwandu hwakakwana hweIPTG? Tora E. coli semuenzaniso.
Mhando yeE. coli BL21 yakagadzirwa nemajini ine positive recombinant pGEX (CGRP/msCT) yakabayiwa muLB liquid medium ine 50μg·mL-1 Amp, uye yakakuriswa husiku hwese pa37°C. Kukuriswa kwataurwa pamusoro apa kwakabayiwa mumabhodhoro gumi e50mL fresh LB liquid medium ine 50μg·mL-1 Amp pachiyero che1:100 chekuwedzera kukura, uye apo kukosha kweOD600 kwaive 0.6~0.8, IPTG yakawedzerwa kuhuwandu hwekupedzisira. I0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mmol·L-1. Mushure mekuiswa kweropa patembiricha imwechete uye panguva imwe chete, 1 mL yemushonga webhakitiriya yakatorwa kubva mairi, uye masero ebhakitiriya akaunganidzwa ne centrifugation ndokuiswa kuSDS-PAGE kuti vaongorore simba rehuwandu hwakasiyana hweIPTG pahuwandu hweprotein, uye vozosarudza huwandu hweIPTG hune huwandu hukuru hweprotein.
Mushure mekuedza, zvichaonekwa kuti huwandu hweIPTG hauna kukura zvakanyanya. Izvi zvinodaro nekuti IPTG ine chepfu chaiyo kumabhakitiriya. Kupfuura huwandu hwesero kuchaurayawo sero; uye kazhinji, tinotarisira kuti kana puroteni inonyunguduka inoratidzwa musero, zviri nani, asi kazhinji kana huwandu hweIPTG hwakanyanya, huwandu hwakawanda hwekubatanidzwa huchaumbwa. Muviri, asi huwandu hwepuroteni inonyunguduka hwakaderera. Naizvozvo, huwandu hwakakodzera hweIPTG kazhinji hausi hukuru huri nani, asi huwandu hwakaderera.
Chinangwa chekusimudzira nekurima mhando dzemajini dzakagadzirwa nemajini ndechekuwedzera goho reprotein inotarwa uye kuderedza mitengo. Kuonekwa kwegene rinotarwa hakungokanganisirwi nezvinhu zvemhando yacho chete uye expression plasmid, asiwo nezvimwe zvinhu zvekunze, zvakaita sekuwanda kweinducer, tembiricha inotarwa uye nguva inotarwa. Saka, kazhinji, protein isingazivikanwe isati yaburitswa nekucheneswa, zvakanaka kudzidza nguva inotarwa, tembiricha uye huwandu hweIPTG kuitira kusarudza mamiriro akakodzera uye kuwana mhedzisiro yakanaka yekuyedza.
Nguva yekutumira: Zvita-31-2021