Part:BBa_K4156103

pLldR-Bxb1-φ174E

pLldR-Bxb1-φ174E is a biobrick that expresses the cleavage gene φ174E by pLldR-Bxb1 co-control.

Usage and Biology

We designed pLldR-Bxb1-φ174E to test the expression efficiency of φ174E under the control of a logic gate linking the pLldR 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 lactate-sensing promoter followed by the amplification genes Switch and mRFP. Fig 1 indicates the lactate (plldR) inducing reporter after the addition of the lysis gene φ174E in induced and non-induced .The lower OD600 values indicate better lysis of the bacteria. Fig 1, it can be seen that the OD600 value becomes lower with increasing lactic acid concentration, and the OD value tends to a more stable state after 20 hour, indicating that our constructed strain can respond well to the tumor environment.

Fig 2 indicates the fluorescence intensity of lactate (plldR) induced reporters under induced and non-induced conditions after the addition of lysis gene φ174E. Fig 2, the fluorescence intensity showed an increasing trend with increasing lactate concentration.

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 lactic acid 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 pLldR+φ174E+Switch+mRFP at different lactate concentrations.

Figure 2: The fluorescence intensity over time by the CR reporter consisting of pLldR +φ174E Switch+ mRFP at different lactate concentrations.

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

To further obtain the lysis-growth curve, we shortened the assay time to 5 min a measurement . Fig 6, Changes in OD600 of lactate (plldR)-induced reporter under induced and non-induced conditions. The results indicate that the lysis-growth curve is a dynamic function.

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

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 plldR-controlled CR were tested by measuring the lactate concentration after collecting the cell supernatant every 12 hours and using this sample as the medium; in Fig 7, as the lactate concentration in the culture increased, more bacteria were lysed and the OD600 values decreased accordingly. Fig 8, the fluorescence response profile was irregular. The results indicate that CR reporters can respond in cell culture medium.

Figure 5:ig The OD600 values of plldR-controlled CR based on the lactate concentration of CT26 cell medium samples.

Figure 6:The fluorescence response of plldR-controlled CR based on the lactate concentration 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 7
  Illegal NheI site found at 30
  Illegal NheI site found at 1479
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1753
  Illegal XhoI site found at 1840
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 614
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2587