I-IPTG (isopropyl-β-D-thiogalactoside) iyi-analogue ye-β-galactosidase substrate, engakwazi ukukhiqizwa kalula. Ngaphansi kokufakwa kwe-IPTG, i-inducer ingakha i-complex ne-repressor protein, Ukuze ukwakheka kwe-repressor protein kushintshwe, ukuze ingakwazi ukuhlanganiswa ne-target gene, futhi i-target gene ivezwe kahle. Ngakho-ke ukuhlushwa kwe-IPTG kufanele kunqunywe kanjani ngesikhathi sokuhlolwa? Ingabe okukhulu kungcono?
Okokuqala, ake siqonde isimiso sokungeniswa kwe-IPTG: I-lactose operon (isici) se-E. coli iqukethe izakhi zofuzo ezintathu, i-Z, Y, kanye ne-A, ezifaka i-β-galactosidase, i-permease, kanye ne-acetyltransferase, ngokulandelana. I-lacZ ifaka i-lactose ku-glucose kanye ne-galactose, noma ibe yi-allo-lactose; i-lacY ivumela i-lactose emvelweni ukuthi idlule kulwelwesi lweseli bese ingena esitokisini; i-lacA idlulisela iqembu le-acetyl kusuka ku-acetyl-CoA iye ku-β-galactoside, okubandakanya ukususa umphumela we-Toxic. Ngaphezu kwalokho, kukhona ukulandelana kokusebenza kwe-O, ukulandelana kokuqala kwe-P kanye ne-gene elawulayo I. Ikhodi yezakhi zofuzo i-I iyiprotheni ecindezelayo engabopha endaweni ye-O yokulandelana komsebenzi, ukuze i-operon (i-meta) icindezelwe futhi icinywe. Kukhona futhi indawo yokubopha yesayithi lokubopha i-catabolic gene activator protein-CAP phezulu kokulandelana kokuqala kwe-P. Ukulandelana kwe-P, ukulandelana kwe-O kanye nesayithi lokubopha le-CAP ndawonye kwakha indawo elawulayo ye-lac operon. Izakhi zofuzo ezibhala ikhodi zama-enzyme amathathu zilawulwa yisifunda esifanayo sokulawula ukuze kufezwe ukubonakaliswa okuhlelekile kwemikhiqizo yezakhi zofuzo.
Uma ingekho i-lactose, i-lac operon (i-meta) isesimweni sokucindezelwa. Ngalesi sikhathi, i-lac repressor evezwa yi-I sequence ngaphansi kokulawulwa kwe-PI promoter sequence ibopha ku-O sequence, okuvimbela i-RNA polymerase ekubopheni ku-P sequence futhi kuvimbele ukuqala kokubhalwa; lapho i-lactose ikhona, i-lac operon (i-meta) ingadalwa. Kulesi simiso se-operon (i-meta), i-inducer yangempela akuyona i-lactose ngokwayo. I-Lactose ingena esitokisini futhi iqhutshwa yi-β-galactosidase ukuze iguqulwe ibe yi-allolactose. Lesi sakamuva, njenge-inducer molecule, sibopha ku-repressor protein futhi sishintsha ukwakheka kweprotheni, okuholela ekuhlukaniseni kweprotheni ye-repressor ku-O sequence kanye nokubhalwa. I-Isopropylthiogalactoside (IPTG) inomphumela ofanayo ne-allolactose. Iyi-inducer enamandla kakhulu, engagaywa ngamagciwane futhi izinzile kakhulu, ngakho-ke isetshenziswa kabanzi emalabhorethri.
Unganquma kanjani ukuhlushwa okuhle kwe-IPTG? Thatha i-E. coli njengesibonelo.
Uhlobo lwe-E. coli BL21 oluyi-genetic engineering oluqukethe i-positive recombinant pGEX (CGRP/msCT) lufakwe ku-LB liquid medium equkethe i-50μg·mL-1 Amp, futhi lwakhuliswa ubusuku bonke ku-37°C. I-culture engenhla ifakwe emabhodleleni ayi-10 e-50mL fresh LB liquid medium equkethe i-50μg·mL-1 Amp ngesilinganiso esingu-1:100 sokukhulisa i-culture, kwathi lapho inani le-OD600 lingu-0.6~0.8, i-IPTG yengezwa ekugxilweni kokugcina. Lingu-0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mmol·L-1. Ngemva kokufakwa ekushiseni okufanayo nangesikhathi esifanayo, kwathathwa i-1 mL yesisombululo sebhaktheriya kuyo, kwathi amaseli ebhaktheriya aqoqwa nge-centrifugation futhi afakwa ku-SDS-PAGE ukuze kuhlaziywe ithonya lamazinga ahlukene e-IPTG ekubonakalisweni kwamaprotheni, bese kukhethwa amazinga e-IPTG anokubonakaliswa okukhulu kwamaprotheni.
Ngemva kokuhlolwa, kuzotholakala ukuthi ukuhlushwa kwe-IPTG akukukhulu ngangokunokwenzeka. Lokhu kungenxa yokuthi i-IPTG inobuthi obuthile kumagciwane. Ukudlula ukuhlushwa kuzobulala neseli; futhi ngokuvamile, sithemba ukuthi uma iphrotheni encibilikayo ivezwa kakhulu esitokisini, kungcono, kodwa ezimweni eziningi lapho ukuhlushwa kwe-IPTG kuphezulu kakhulu, kuzokwakhiwa inani elikhulu lokufakwa. Umzimba, kodwa inani lephrotheni encibilikayo lehlile. Ngakho-ke, ukuhlushwa kwe-IPTG okufanele kakhulu kuvame ukungabi kukhulu kungcono, kodwa lapho ukuhlushwa kuphansi.
Inhloso yokungenisa nokutshala izinhlobo ezakhiwe ngezakhi zofuzo ukwandisa isivuno seprotheyini eqondiwe nokunciphisa izindleko. Ukuvezwa kwe-gene eqondiwe akugcini nje ngokuthintwa yizici zohlobo kanye ne-plasmid yokubonakaliswa, kodwa futhi nezinye izimo zangaphandle, njengokuhlushwa kwe-inducer, izinga lokushisa lokungeniswa kanye nesikhathi sokungeniswa. Ngakho-ke, ngokuvamile, ngaphambi kokuba iphrotheyini engaziwa ivezwe futhi ihlanzwe, kungcono ukutadisha isikhathi sokungeniswa, izinga lokushisa kanye nokuhlushwa kwe-IPTG ukuze ukhethe izimo ezifanele futhi uthole imiphumela emihle kakhulu yokuhlola.
Isikhathi sokuthunyelwe: Disemba 31-2021