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.
Characterization
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 2, the fluorescence intensity under normoxic conditions was very low, while the fluorescence intensity under hypoxic conditions increased significantly after 8h.
Fig3 is 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.
To further obtain the lysis-growth curve, we shortened the assay time to 5 min a measurement . Fig4, 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.
Next, we tested the constructed CR reporters using CT26 cell cultures. In Fig5, 6, 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 5 and 6, 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. Fig5, As the pH decreased, more bacteria were lysed and the OD600 values showed a decreasing trend. Fig6, the fluorescence intensity shows an increasing trend as the pH decreases. The results indicate that CR reporters can respond in cell culture medium.
Lactate (plldR) and pH (pPepT)Induced promoter-controlled effector engineered strain co-incubated with RKO cells
Figure 7 shows the RKO cell activity after incubation of each strain in fresh DMEM medium, normoxic conditions(OD=0.6, 30 μl, 3 hours). It can be seen that the RKO relative viability of the experimental groups with the addition of the effector strains in the fresh culture medium did not change significantly compared to the WT group, except for the plac+HlyE positive control. Figure 8 shows the RKO cell activity of each strain after incubation in 3 day DMEM medium, normoxic conditions. It can be concluded that in the 3 day DMEM medium, due to the accumulation of metabolites such as cellular lactate, the lactate promoter and pH promoter were activated in the engineered strains and started to synthesize therapeutic proteins, resulting in a decrease in the relative viability of RKO compared to the WT group, especially in the pLldR+switch+HlyE and pCadC+switch+HlyE groups with the addition of the amplified gene switch. switch+HlyE group with the addition of the amplifying gene switch significantly reduced the RKO relative viability. In contrast, the decrease in RKO relative viability in the pLldR+φ174E+switch+HlyE group and pCadC+φ174E+switch+HlyE group was not significant, probably due to the decrease in the number of bacteria and the decrease in the number of synthesized therapeutic proteins by the addition of lysis genes.
Coincubation of different doses of effector engineered strains (OD=0.6) with RKO cells
Figure 9 shows the RKO cell activity after incubation with different doses of plldR and pCadC control effector strains in 3 day DMEM medium, normoxic conditions. The RKO cell activity decreased with increasing doses of effector strains.
30 μl effector engineered strains (OD=0.6) were co-incubated with RKO cells for different times
Figure 10 shows the RKO cell activity after incubation of plldR and pCadC control effector strains for different times under 3 day DMEM medium, normoxic conditions. It can be seen that the RKO cell activity decreased with the increase of co-incubation time.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 368
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 642
Illegal XhoI site found at 729 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1476
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