Part:BBa_K4307037

J32100-AffiPmrB-PmrA-PmrC-T7 core-T3 sigma-pT3-EGFP

This part is improved from our AffiPmrB/A/C part (BBa_K4307042). The fluorescence signal output by the engineered bacteria with AffiPmrB/A/C was very weak. Since our goal was to observe the fluorescence signal with naked eyes, we introduced the T7-T3 cascade amplifier into our Affi-Pmr system to amplify the output fluorescence signal output. T7-core-T3-sigma system is developed from T7 and T3 bacterial phage, the core enzyme of T7 RNA polymerase could interact with the T3 sigma transcription factor, precisely promote the expression of pT3 promoter. We put the T3 sigma under PmrC, thus the signal produced by AffiPmrB/A/C would be amplified and showed as the EGFP fluorescence intensity.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 896
  Illegal BamHI site found at 2161
  Illegal BamHI site found at 2331
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 5837
  

Characterization

The following figure demonstrates our successful construction.

Figure 1: The construction results of J32100-AffiPmrB-PmrA-PmrC-T7 core-T3 sigma-pT3-EGFP.

Fluorescence assay was done to characterize the biobrick.

The engineered DH5α was incubated with different concentration of IgG. After 10h incubation, the EGFP fluorescence intensity of the DH5α was detected by microplate reader:

Figure 2: Fluorescence spectrophotometry of Affi-PmrB/A-amplifier system. We can see that the EGFP fluorescence/OD600 is much higher than AffiPmrB/A/C system (both induced and uninduced), while the bacteria induced by 200μM IgG has the highest EGFP fluorescence intensity, which is about 40% higher than the non-induced bacteria.

Conclusion

The T7-T3 amplifier system helps confirming the effectiveness of AffiPmrB/A/C system, but it also shows that the AffiPmrB/A/C system is a weak expressing system, as the high background level shows above.