Part:BBa_K4156105
pLldR-TP901-φ174E
pLldR-TP901-φ174E is a composite part that express lysis gene φ174E.
Usage and Biology
We designed pLldR-TP901-φ174E to test the expression efficiency of φ174E under the control of a logic gate linking the pLldR ( BBa_K4156101 ) and the serine integrase TP901( BBa_K4156112 ). We will validate the function of this biobrick by measuring the viability of the bacteria.
Characterization
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( BBa_K4156114 ). 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. 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. 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.
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.
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.
Lactate (pLldR) and pH (pCadC)Induced promoter-controlled effector engineered strain co-incubated with RKO cells
We linked pLldR-TP901--φ174E to XOR gate-HlyE （ BBa_K4156119 ) for validation of treatment viability.
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
We linked pLldR-TP901--φ174E to XOR gate-HlyE （ BBa_K4156119 ) for validation of treatment viability.
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
We linked pLldR-TP901--φ174E to XOR gate-HlyE （ BBa_K4156119 ) for validation of treatment viability.
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.
Western blot
To verify the extracellular secretion of HlyE, we constructed an AE strain by fusing his tag at the C-terminus of HlyE. Then, the AE strain (HlyE with his tag) was inoculated in 50 ml of LB medium containing the corresponding antibiotics and cultured overnight at 37 °C. Then, 5 ml of the culture was centrifuged and the supernatant was collected. The supernatant was concentrated using the TCA precipitation method (25% TCA, -20°C, 1h) to isolate the total protein. Finally, the expression of HlyE was detected by western blot. The results showed that the constitutive promoter could secrete HlyE under both inducible and non-inducible conditions, while the lactate (plldR), pH (pCadc) and hypoxia (pPepT) inducible reporters could only secrete HlyE under inducible conditions and not under non-inducible conditions. indicated that our constructed AE strain could well cope with environmental induction and secrete HlyE in the tumor microenvironment It was shown that our AE strain could respond well to environmental induction and secrete HlyE in the tumor microenvironment, thus killing cancer cells without harming other normal cells.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 614
Illegal AgeI site found at 2743 - 1000COMPATIBLE WITH RFC[1000]
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