Part:BBa_K3885342

P70-σ28-P28-tetR

Under the influence of the P70 promoter, the transcription factor σ28 is expressed, which activates the P28 promoter to express the repressor tetR and represses the expression of downstream genes

Usage and Biology

Tet Repressor (tetR) can express a repressor protein, which can binds to the tetO site and thus blocks deGFP. We constructed this part and combined the P28-tetO-deGFP part to test the inhibitory effect of tetR on deGFP. At the same time, this part can be combined with CRISIPR, using tetR as a target gene, and indirectly feedback the cutting efficiency of Cas9 through the inhibitory effect of fluorescence, and transmit information through visible fluorescence.

Characterization

This part mainly combines with tetO to verify whether it can successfully inhibit the expression of deGFP. Through electroporation of BW25113 and plasmids containing P28-tetO-deGFP and plasmids containing P70a-σ28-P28-tetR, it was verified whether tetR could successfully inhibit the expression of deGFP. The experimental results show that the fluorescence results of the group containing this part (Figure 1 b) are significantly different from those of the control group (Figure 1 a), which proves that this part functioned as intended, tetR was able to normally express the intensity of the repressor protein inhibiting deGFP. Three groups of BW25113; BW25113+P28a-tetO-deGFP plasmid; BW25113+P28-tetO-deGFP plasmid+P70-σ28-P28-tetR plasmid were electroporated respectively; after coating, the bacteria were picked and cultured and the fluorescence was measured, and the results showed that BW25113 was transferred into P28a-tetO-deGFP plasmid and P70-σ28-P28-tetR plasmid, its fluorescence is significantly weaker than that of BW25113+P28-tetO-deGFP plasmid, which proves that this part can function.

Design Page

The part was designed to bind to the P28-tetO-deGFP, and the intracellular verification was conducted through an electrotransfer. The inhibitory effect of tetR on deGFP was qualitatively determined, and it was taken as the target gene of Cas9 in a Cell-Free system. The fluorescence intensity before and after Cas9 binding igRNA was quantitatively determined, and the concentration of our RNA markers was quantitatively determined by the fluorescence intensity.

References

Akiko Kashiwagi,Takahiro Sakurai,Saburo Tsuru,Bei-Wen Ying,Kotaro Mori,Tetsuya Yomo. Construction of Escherichia coli gene expression level perturbation collection[J]. Journal of Bioscience and Bioengineering,2009,108:

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 487
  Illegal SapI.rc site found at 512
  Illegal SapI.rc site found at 668