Ko te IPTG (isopropyl-β-D-thiogalactoside) he rite ki te matū β-galactosidase, he mea tino kaha ki te whakaoho. I raro i te whakaoho o te IPTG, ka taea e te kaiwhakaoho te hanga i tētahi matatini me te pūmua aukati, kia rerekē ai te āhua o te pūmua aukati, kia kore ai e taea te hono atu ki te ira ūnga, ā, kia pai ai te whakapuakitanga o te ira ūnga. Nō reira, me pēhea te whakatau i te kukū o te IPTG i te wā o te whakamātautau? He pai ake te nui ake?
Tuatahi, kia mārama tātou ki te kaupapa o te whakaoho IPTG: Kei roto i te operon lactose (huānga) o E. coli e toru ngā ira hanganga, Z,Y, me A, e whakakōrero ana i te β-galactosidase, permease, me te acetyltransferase. Ka whakawairakau a lacZ i te lactose hei glucose me te galactose, hei allo-lactose rānei; ka taea e lacY te lactose i roto i te taiao kia haere mā roto i te kiriuhi pūtau ka tomo ki te pūtau; ka whakawhiti a lacA i te rōpū acetyl mai i te acetyl-CoA ki te β-galactoside, e uru ana ki te tango i te pānga paitini. Hei tāpiri, he raupapa mahi O, he raupapa tīmatanga P me te ira whakahaere I. Ko te waehere ira I he pūmua pehi ka taea te here ki te tūranga O o te raupapa kaiwhakahaere, kia pehia ai te operon (meta) ka whakawetohia. He pae here hoki mō te pae here pūmua whakahohe ira catabolic-CAP i mua o te raupapa tīmatanga P. Ko te raupapa P, te raupapa O me te pae here CAP e hanga tahi ana i te rohe whakahaere o te operon lac. Ka whakahaeretia ngā ira waehere o ngā whākōkī e toru e te rohe whakahaere kotahi kia tutuki ai te whakaaturanga taunga o ngā hua ira.
I te korenga o te lactose, kei roto te operon lac (meta) i te āhua pehi. I tēnei wā, ka herea te kaiārai lac e whakaatuhia ana e te raupapa I i raro i te mana o te raupapa whakatairanga PI ki te raupapa O, e ārai ana i te here o te RNA polymerase ki te raupapa P, ā, e ārai ana i te tīmatanga o te tuhi; ina kitea te lactose, ka taea te whakaoho i te operon lac (meta). I roto i tēnei pūnaha operon (meta), ehara i te lactose tonu te kaiārai tūturu. Ka tomo te lactose ki te pūtau, ā, ka whakakorikoria e te β-galactosidase kia huri hei allolactose. Ko te mea whakamutunga, hei ngota kaiārai, ka herea ki te pūmua kaiārai, ā, ka whakarerekē i te āhua o te pūmua, ka arahi ki te wehenga o te pūmua kaiārai mai i te raupapa O me te tuhi. He rite tonu te pānga o te Isopropylthiogalactoside (IPTG) ki te allolactose. He kaiārai tino kaha, kāore e taea te whakapūngao e ngā huakita, ā, he tino pumau, nō reira he whānui te whakamahinga i roto i ngā taiwhanga.
Me pēhea te whakatau i te kukū tino pai o te IPTG? Hei tauira, me tango te E. coli.
Ko te momo E. coli BL21 i hangaia i te ira kei roto ko te pGEX recombinant pai (CGRP/msCT) i werowerohia ki roto i te wai LB kei roto ko te 50μg·mL-1 Amp, ā, i whakatipuria mō te pō i te 37°C. Ko te ahurea i runga ake nei i werowerohia ki roto i ngā pounamu 10 o te wai LB hou 50mL kei roto ko te 50μg·mL-1 Amp i te ōwehenga 1:100 mō te ahurea whakawhānui, ā, i te wā i 0.6~0.8 te uara OD600, i tāpirihia te IPTG ki te kukū whakamutunga. Ko te 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mmol·L-1. I muri i te whakaurunga i te pāmahana kotahi me te wā kotahi, ka tangohia te 1 mL o te otinga huakita mai i roto, ā, ka kohia ngā pūtau huakita mā te centrifugation ka tukuna ki te SDS-PAGE hei tātari i te awe o ngā kukū IPTG rerekē ki te whakaaturanga pūmua, kātahi ka tīpakohia te kukū IPTG me te whakaaturanga pūmua nui rawa atu.
I muri i ngā whakamātautau, ka kitea kāore te kukū o te IPTG i te tino nui. Nā te mea he paitini te IPTG ki ngā huakita. Mā te nui rawa atu i te kukū ka mate te pūtau; ā, i te nuinga o te wā, ko te tumanako ka pai ake te nui o te pūmua wairewa i roto i te pūtau, engari i roto i te nuinga o ngā wā ka nui rawa te kukū o te IPTG, ka nui te whakaurunga ka puta. Tinana, engari ka heke te nui o te pūmua wairewa. Nō reira, ko te kukū IPTG tino pai ehara i te mea ko te nui ake ko te pai ake, engari ko te iti iho o te kukū.
Ko te kaupapa o te whakaoho me te whakatipu i ngā momo ira kua hangaia he ira, he whakanui ake i te hua o te pūmua ūnga me te whakaiti i ngā utu. Ehara i te mea ko ngā āhuatanga o te momo me te plasmid te mea anake e pāngia ana e te whakaaturanga o te ira ūnga, engari ko ētahi atu āhuatanga o waho anō hoki, pērā i te kukū o te kaiwhakaoho, te pāmahana whakaoho me te wā whakaoho. Nō reira, i te nuinga o te wā, i mua i te whakaputanga me te purenga o tētahi pūmua kāore i te mōhiotia, he mea pai kia akohia te wā whakaoho, te pāmahana me te kukū IPTG kia taea ai te whiriwhiri i ngā āhuatanga e tika ana, kia whiwhi ai i ngā hua whakamātautau pai rawa atu.
Wā tuku: Hakihea-31-2021