Team:ASTWS-China/Measurement

• Home
• Project▾
  • Description
  • Design
  • Applied Design
• Experiment▾
  • Results
  • Demonstrate
  • Improve
  • Notebook
  • Protocol
  • Safety
• Measurement
• Human Practice▾
  • Human Practices
  • Public Engagement
  • Collaborations
• Parts
• Model
• Team▾
  • Team Members
  • Attributions

Measurement

---

For characterization part, we deeply agree with what Jacob Beal said, "Measurement is fundamentally an act of communication." Quantification and reproducibility is the basis and another important thing of bioengineering.

In this summer, our team recharacterized the biobrick BBa_K2826005, a device to detect the copper ion. Considering the universal use of GFP in iGEM competition, GFP reporter was used to characterize the performance of this copper-sensitive promoter.

We constructed the plasmids pSB1C3-pCopA-Riboj (BBa_K2826005) and pSB1C3-GFP (BBa_E0840) and finally obtained the working plasmid pSB1C3-pCopA-Riboj-GFP via enzyme digestion and ligation. To verify the function of pCopA promoter, the bacteria containing the working plasmid was cultured at different copper concentrations for 10 hours. The fluorescence of GFP was measured every 2 hours. When culturing for 0 ~ 8 hours, the result showed that the GFP fluorescence value is positively correlated with the copper concentration (Figure 1) as well as the culturing time. As shown in Figure 1A, during 8 to 10 hours, the growth of fluorescence value tended to be flat.

Figure 1 Curve of GFP fluorescence intensity under different copper concentrations after culturing (A) 0~10 hours and (B) 8 hours.

The E. Coli DH5α containing the working plasmids were growing at the copper concentration of 0, 5 and 50 mg/L. The representative photo was shown in Figure 2. Clear expression was visible after collecting the bacteria by centrifugation. The result also showed that the fluorescence intensity increased with the increase of copper concentration (Figure 2).

Figure 2 Results of E. Coli DH5α after culturing overnight under different copper concentrations.

Conclusion

To sum up, this summer, we recharacterized the performance of copper-sensitive promoter in BBa_K2826005 using the common-used reporter protein, GFP. It is proven that the biobrick BBa_K2826005 is reproducibility and functioning stably under the copper concentration of 0-125 mg/L. The fluorescence value is positively correlated with the copper concentration and the culturing time before 8 hours. At 8 to 10 hours, the growth of fluorescence value tended to be flat. The supplementing experimental data have been submitted to the Part’s registry page (BBa_K2826005) and our WIKI page. We hope that our experiences can give others a basis for comparison and reference.