Part:BBa_K3152008

copper sensor2

This part is the improvement of part BBa_K2826008,a lysis part has been added to control the death of the host cell.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 898
  Illegal AgeI site found at 1010
  
• 1000
  COMPATIBLE WITH RFC[1000]

Description & Design

This part is the improvement of part BBa_K2826008, which consisted of the copper ion detecting promoter (pCopA) and a reporter (RFP). To solve the problems of spreading genetic-engineered strains in the environment, we designed the improved system which could lead the strains death when IPTG is added to the culture medium. We added a composite part consisted of alacI regulated promoter (BBa_R0010) and a lysis gene (BBa_K117000) following the existed part. The composition of part BBa_K3152008 is shown in Figure 1.

Figure 1 Composition of part BBa_K3152008

Results and Discussion

We first constructed the lacI regulated promoter (BBa_R0010)+RBS+lysis gene (BBa_K117000) fragment. The results are shown in Figure 2 (Left & Middle). The we used pSB1C3-RFP as the backbone and add the fragment consisting of lacI promoter+RBS+lysis gene into the plasmid pSB1C3-RFP. After culturing on the LB agar plates containing Cu2+ (50mg/mL), transformants were chosen and the plasmids were extracted and verified by enzyme digestion. The gel-electrophoresis result in Figure 2 (Right) showed the correct size (1.6kb). The DNA sequencing result afterward was also proven to be correct.

Figure 2 PCR results of the fragment of lacI promoter+RBS+lysis gene (Left); gel-electrophoresis results of fragments after digesting with restriction enzymes (Middle); gel-electrophoresis of the plasmids digesting with EcoRI and PstI (Right).

To testify the function of our improved part, strains were cultured in LB broth containing Cu2+. As shown in Figure 3A, with the extension of culture time, the OD600 value of both sample and control group increased. When IPTG was added at 4 hours, the OD600 value of sample group decreased meanwhile the control group was not affected. The consequence of Figure 3B also supported the result. It showed that the red fluorescence increased with the extension of culture time before adding IPTG into the culture, and the red fluorescence decreased after adding IPTG, indicating that the lysis gene was successfully expressed and led to the death of host cells.

Figure 3 Results of the OD600 values (A) and red fluorescent values (B) at different culture time.

---

Conclusion

The design would be used as a well-functioning device to detect Cu2+ ion in medium. This improved system is repeatable and effective. With the existence of lysis gene, the growth of bacteria was effectively inhibited. The culture conditions and the concentration of IPTG could be optimized in the future work. It is expected to control the death of bacteria whenever we need by adding IPTG into the culture medium, which could avoid the spread of genetic-engineered strains in the environment and prevent hazards to the environment.