Part:BBa_K4156107

pPepT-Bxb1-φ174E

pPepT-Bxb1-φ174E is a composite part that express lysis gene φ174E, constructed with hypoxia-sensing promoter pPepT and serine integrase Bxb1.

Usage and Biology

We designed pPepT-Bxb1-φ174E to test the expression efficiency of φ174E under the control of a logic gate linking the pPepT and Bxb1 We will validate the function of this biobrick by measuring the viability of the bacteria.

In vitro characterization and data analysis of the reported strains withφ174E

We constructed the lysis reporter CR by adding the pH-sensing promoter followed by the amplification genes Switch and mRFP. Fig 1 indicates the hypoxia (pPepT) inducing reporter after the addition of the lysis geneφ174E in induced and non-induced.The lower OD600 values indicate better lysis of the bacteria. Fig 1, the OD600 under anoxic conditions was lower than that under normoxic conditions, indicating that our constructed strain can respond well to the tumor environment. Fig 2 indicates the fluorescence intensity of hypoxia (pPepT) induced reporter under induced and non-induced conditions after the addition of lysis gene φ174E. Fig 2, the fluorescence intensity under normoxic conditions was very low, while the fluorescence intensity under hypoxic conditions increased significantly after 8h. Fig 3-5 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 anoxic 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 pPepT+φ174E+Switch+mRFP under hypoxic and normoxic conditions

Figure 2: The fluorescence intensity over time by the CR reporter consisting of pPepT+φ174E+Switch+mRFP under hypoxic and normoxic conditions.

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 . Fig 6, change in OD600 of hypoxia (pPepT)-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 at different pH values.

Next, we tested the constructed CR reporters using CT26 cell cultures. In Fig 7 and 8, CT26 cells were cultured for 5 consecutive days, and the OD600 values and fluorescence response of the pPepT-controlled CR were tested. Fig 7 and 8, OD600 values and fluorescence response of pPepT-controlled CR tested using the above cell culture medium. Fig 7, OD600 values under anaerobic conditions were significantly smaller than those under normoxic conditions, and Fig 8 fluorescence values under anaerobic conditions were higher than those under normoxic conditions. The results indicate that CR reporters can respond in cell culture medium.

Figure 7:ig 7. The OD600 values of pPepT-controlled CR based on CT26 cell medium samples under hypoxic and normoxic conditions.

Figure 8:The fluorescence response of pPepT-controlled CR based on CT26 cell medium samples under hypoxic and normoxic conditions.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 360
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 634
  Illegal XhoI site found at 721
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1468