Part:BBa_K2165004:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Characterization by iGEM2017_Tianjin

Based on the CUP1 promoter (BBa_K2165004) provided by iGEM16_Washington, we constructed a biosensor with yEmRFP. To characterize this biosensor, strains of S. cerevisiae BY4742 containing the plasmid with an initial OD₆₀₀ of 0.1 were grown for 24 hours in SC-URA medium at 30 degrees Celsius, and then were induced with copper sulfate. Samples in different copper concentration were tested with fluorescent microplate reader after 1, 6, 12, and 24 hours. This protocol was based on the experience used by Waterloo and Washington iGEM teams and amended by our team.

Fig 1.The fluorescence intensity of CUP1p-yEmRFP biosensor with different Cu concentration induced

Figure 1 showed the relationship between fluorescence intensity with induction time and Cu²⁺ concentration. With 0.1 mM CuSO₄ induced, the fluorescence intensity is 2 times over a control with no induction at 1 hour. As time went on, the fluorescence intensity slightly reduced. Moreover, as the Cu²⁺ concentration increased, the fluorescence intensity decreased, and when the concentration reached 1 mM, the intensity was close to the control group. This might be due to the higher copper ion concentration influences the transcription, expression and even growth of yeast.

Improvement by iGEM2017_Tianji

Based on this BioBrick, we redesigned the part sequence provided by iGEM16_Washington. We deleted irrelevant bases on the two ends of this promoter and retained the core sequence. We retained 5 ACE1/CUP1 binding sites, 2 TATA boxes, and one initiation element in the promoter. The complex of ACE1 and copper ions will bind the promoter, which causes the activation of CUP1 promoter with TATA boxes' help. In this way, this promoter played its key role with less bases.

Visit BBa_K2407000 to see more information.

Fig 2.Structure of redesigned CUP1 promoter used in our project, based on BBa_K2165004

Actually, we built some new parts based on CUP1 promoter by error-prone PCR. Visit BBa_K2407013, BBa_K2407014, BBa_K2407015, and BBa_K2407016 to get more information about characterization.

Applications of BBa_K2165004

A Cu²⁺ Biosensor made by iGEM2017_Tianjin

We combined BBa_K2165004 and BBa_K2407012 to build a Cu²⁺ Biosensor. The characterization can be found above.

If you want see more information, please visit: 2017.igem.org/Team:Tianjin/Demonstrate

User Reviews