Part:BBa_K3525001

T7 pro-His-ER sensor-His-T7 ter

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 923
  Illegal BglII site found at 1420
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1845
  
• 1000
  COMPATIBLE WITH RFC[1000]

BBa_K3525001 contains BBa_K3525000, a sensor, used to detect endocrine-disrupting chemicals (EDCs).

Contribution

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.

figure 1

Engineering Success

We plan to test the functionality of our constructed ER Senor in presence of estrogen. To accomplish this functional test, we made the following experiments: 1. 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 2

Figure 2: 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 2. To confirm the color reaction of lacZ reporter protein contained in our ER sensor, we tested and confirmed the color reaction of ONPG and lacZ protein. The corresponding results were listed as follows:

figure 3

Figure 3: 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. According to Figure 3, the color of sample 2 and sample 3 were different. It proves that 1) The lacZ protein reporter in our ER Sensor works; 2) The bright yellow color reaction of ONPG and lacZ is more suitable for visual inspection than the blue color reaction of x-gal.