Difference between revisions of "Part:BBa K4156100"

(Lactate (plldR) and pH (pPepT)Induced promoter-controlled effector engineered strain co-incubated with RKO cells)
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==Lactate (plldR) and pH (pPepT)Induced promoter-controlled effector engineered strain co-incubated with RKO cells==
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==Lactate (pLldR) and pH (pCadC)Induced promoter-controlled effector engineered strain co-incubated with RKO cells==
 
We linked pCadC-TP901-φ174E to XOR gate-HlyE ( <html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156119"> BBa_K4156119 </a></html>) for validation of treatment viability
 
We linked pCadC-TP901-φ174E to XOR gate-HlyE ( <html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156119"> BBa_K4156119 </a></html>) for validation of treatment viability
  

Revision as of 01:40, 12 October 2022


pCadC-TP901-φ174E

pCadC-TP901-φ174E is a composite part that express lysis gene φ174E, constructed with pH-sensitive promoter pCadC and serine integrase TP901.


Usage and Biology

We designed pCadC-TP901-φ174E to test the expression efficiency of φ174E under the control of a logic gate linking the pCadC ( BBa_K4156076 ) and the serine integrase TP901 ( BBa_K4156087 ). 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 with φ174E

We constructed the lysis reporter CR by adding pH-sensing promoter followed by the amplification genes Switch and mRFP.( BBa_K4156118 )

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.The fluorescence intensity showed an upward trend with decreasing pH, and the fluorescence intensity under normoxic conditions was very low, while the fluorescence intensity under hypoxic conditions increased significantly after 8h.

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

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

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.

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

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.

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Figure 4: 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 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 Fig 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.

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Figure 5: The OD600 values of pCadC-controlled CR based on the pH of CT26 cell medium samples.

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Figure 6: The fluorescence response of pCadC-controlled CR based on the pH of CT26 cell medium samples.

Lactate (pLldR) and pH (pCadC)Induced promoter-controlled effector engineered strain co-incubated with RKO cells

We linked pCadC-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.

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Figure 7:The activity of RKO cells after incubation with each strain (OD=0.6, 30 μl, 3 hours) in fresh DMEM medium, normoxic conditions.
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Figure 8:The activity of RKO cells after incubation with each strain (OD=0.6, 30 μl, 3 hours) in 3 day DMEM medium, normoxic conditions.

Coincubation of different doses of effector engineered strains (OD=0.6) with RKO cells

We linked pCadC-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.

control
Figure 9:The RKO cell activity after incubation with different doses of plldR and pCadC control effector strains under 3 day DMEM medium, normoxic conditions.

30 μl effector engineered strains (OD=0.6) were co-incubated with RKO cells for different times

We linked pCadC-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.

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Figure 10:The RKO cell activity after incubation of plldR and pCadC control effector strains for different times under 3 day DMEM medium, normoxic conditions..


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1632
  • 1000
    COMPATIBLE WITH RFC[1000]