Part:BBa_K4156098

pCadC-Bxb1-φ174E

pCadC-Bxb1-φ174E is a complex component expressing the cleavage gene φ174E, constructed from the pH-sensitive promoter pCadC and the serine integrase Bxb1.

Usage and Biology

We designed pCadC-Bxb1-φ174E to test the expression efficiency of φ174E under the control of a logic gate coupling the pCadC promoter to Bxb1. We will validate the function of this biobrick by measuring bacterial viability.

In vitro characterization and data analysis of the reported strains with φ174E

We constructed the lysis reporter CR by adding pH-sensing promoter followed by the amplification genes Switch and mRFP. Fig1 indicates pH (pCadc) inducing reporters after the addition of the lysis gene φ174E in induced and non-induced .The lower OD600 values indicate better lysis of the bacteria. Fig1, as the pH decreases, the OD600 value also decreases,indicating that our constructed strain can respond well to the tumor environment.

Fig2 indicates the fluorescence intensity of pH (pCadc) induced reporters under induced and non-induced conditions after the addition of lysis geneφ174E. Fig2 the fluorescence intensity showed an upward trend with decreasing pH, and Fig (F), the fluorescence intensity under normoxic conditions was very low, while the fluorescence intensity under hypoxic conditions increased significantly after 8h.

Fig3-5 are the OD600 of wild-type 1917 bacteria under induced and non-induced conditions, and the wild-type bacteria could hardly respond to the induction of pH environments. The results show that CR undergoes lysis under induced conditions, but the cells still produce fluorescence. It indicates that the fitted set of equations for lysis-growth should be a resonance function.

Figure 1: The OD600 values over time by the CR reporter consisting of pCadc+φ174E+Switch+mRFP at different pH values.

Figure 2: Induction of downstream gene mRFP expression over time by the CR reporter consisting of pCadc+φ174E+Switch+mRFP at different pH values.

Figure 3: The OD600 values over time of wild-type 1917 bacteria under induced and non-induced conditions at different lactate concentrations.

Figure 4: The OD600 values over time of wild-type 1917 bacteria under induced and non-induced conditions at different pH values.

Figure 5: The OD600 values over time of wild-type 1917 bacteria under induced and non-induced conditions under hypoxic and normoxic conditions.

To further obtain the lysis-growth curve, we shortened the assay time to 5 min a measurement . Fig6, OD600 changes of pH(pCadc)-induced reporter under induced and non-induced conditions.The results indicate that the lysis-growth curve is a dynamic function.

Figure 6: The OD600 values over time of wild-type 1917 bacteria under induced and non-induced conditions by under hypoxic and normoxic conditions.

Next, we tested the constructed CR reporters using CT26 cell cultures. In Fig7, 8, CT26 cells were cultured for 5 consecutive days, and the OD600 values and fluorescence response of the pCadC-controlled CR were tested by measuring the pH after collecting the cell supernatant every 12 hours and using this sample as the medium; In 7 and 8, the pH level of the above cell culture medium sample was measured and used as the medium to test the OD600 value and fluorescence response of the pCadC-controlled CR. Fig7, As the pH decreased, more bacteria were lysed and the OD600 values showed a decreasing trend. Fig8, the fluorescence intensity shows an increasing trend as the pH decreases. The results indicate that CR reporters can respond in cell culture medium.

Figure 7: The OD600 values of pCadC-controlled CR based on the pH of CT26 cell medium samples.

Figure 8: The fluorescence response of pCadC-controlled CR based on the pH of CT26 cell medium samples.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 368
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 642
  Illegal XhoI site found at 729
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1476