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Composite part BBa_K3525001 contains ER-sensor-S protein coding sequence. It is used to express and purify ER-sensor-S protein. We have carried out molecular biology experiments in a laboratory, and have successfully constructed the designed ER-sensor-S protein coding sequence into the expression vector pET28a by PCR and multi-fragment assembly reaction.
The plasmid pET28a-ER-sensor was transferred into BL21 (DE3) and induced by IPTG. We tested and verified the expression of ER sensor protein by SDS-PAGE.
Figure 1: Expression and characterization of ER sensor protein
1: Negative control, 2: Lysate of ER sensor protein expression strain, 3: Estrogen molecules added into the lysate of ER sensor protein expression strain, 4: Positive control of LacZ expression bacteria lysate
In order to confirm the color reaction of lacZ reporter protein, we tested and confirmed the color reaction of ONPG and lacZ protein.
Figure 2: Color reaction result of ER sensor protein with ONPG in the presence of estrogen
1: Negative control, 2: color reaction of ER sensor protein expression bacteria lysate without estrogen, 3: color reaction of ER sensor protein expression bacteria lysate with estrogen.
We found that the bright yellow color reaction of ONPG and lacZ is more suitable for visual inspection than the blue color reaction of x-gal.
We confirmed that the optimum reaction temperature of ONPG and lacZ was 37 ℃.
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