Part:BBa_K3525005
T7 pro-His-lacZ-His-T7 ter
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Contribution
Composite part BBa_K3525005 contains the coding sequences of LacZ.
Engineering Success
We transformed a pET28a plasmid with lacZ into E. coli BL21 (DE3) and induced lacZ expression by IPTG. After the verification of successful expression of lacZ, we used the HIS tag on lacZ when expressed on pET28a plasmid to purify the lacZ protein by nickel column. We obtained the purified lacZ protein which is about 110KD by elution with 250mm imidazole (Figure 2).
Figure 2: Expression and purification of lacZ protein
1: Negative control, 2: Lysate of lacZ expressing BL21(DE3), 3: precipitation of lacZ expression BL21(DE3), 4: supernatant of lacZ expression BL21(DE3), 5: Nickel column flow-through fluid of lacZ expression BL21(DE3), 6: 15mM imidazole eluent, 7: 250mM imidazole eluent Then we identified the purified lacZ protein by X-gal (Figure 3) and ONPG (Figure 4) respectively. The results showed that both reactions could detect lacZ, and the bright yellow color reaction of ONPG was more obvious from the naked eye. At the same time, we confirmed that the optimum reaction temperature of lacZ with ONPG is 37℃ (Figure 4A). And the reaction can be quantified by detecting the absorbance at 420nm (Figure 4B).
Figure 3: Identification of purified lacZ protein by adding different amount of x-gal
Figure 4: Identification of purified lacZ protein by reacting with ONPG at different temperatures.
A: Color reaction of lacZ with ONPG, B: Absorption values of lacZ and ONPG color reaction at 420nm
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