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| | ===Characterization=== | | ===Characterization=== |
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| | + | We used three different promoters in combination with XOR gate-HlyE to characterize the therapeutic efficacy of bacteria under the control of different promoters and TP901 coupled logic gates, and with the addition of lysis genes. |
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| − | ==Lactate (plldR) and pH (pPepT)Induced promoter-controlled effector engineered strain co-incubated with RKO cells==
| + | Characterization details can be found at： |
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| − | 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.
| + | pCadC-TP901:<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156099"> BBa_K4156099 </a></html> |
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| − | <html> | + | pCadC-TP901-φ174E:<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156100"> BBa_K4156100 </a></html> |
| − | <figure style="text-align:center;"> | + | |
| − | <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/10-1.png" alt="control">
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| − | <figcaption><b>Figure 7:</b>The activity of RKO cells after incubation with each strain (OD=0.6, 30 μl, 3 hours) in fresh DMEM medium, normoxic conditions.</figcaption>
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| − | </figure>
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| − | </html> | + | pLldR-TP901:<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156104"> BBa_K4156104 </a></html> |
| − | <html> | + | |
| − | <figure style="text-align:center;">
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| − | <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/10-2.png" alt="control">
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| − | <figcaption><b>Figure 8:</b>The activity of RKO cells after incubation with each strain (OD=0.6, 30 μl, 3 hours) in 3 day DMEM medium, normoxic conditions.</figcaption>
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| − | </figure>
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| − | </html> | + | |
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| − | ==Coincubation of different doses of effector engineered strains (OD=0.6) with RKO cells==
| + | pLldR-TP901-φ174E:<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156105"> BBa_K4156105 </a></html> |
| − | 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.
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| − | <html> | + | pPepT-TP901:<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156108"> BBa_K4156108 </a></html> |
| − | <figure style="text-align:center;"> | + | |
| − | <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/10-3.png" alt="control">
| + | pPepT-TP901-φ174E:<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156109"> BBa_K4156109 </a></html> |
| − | <figcaption><b>Figure 9:</b>The RKO cell activity after incubation with different doses of plldR and pCadC control effector strains under 3 day DMEM medium, normoxic conditions.</figcaption>
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| − | </figure>
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| − | </html> | + | |
| − | ==30 μl effector engineered strains (OD=0.6) were co-incubated with RKO cells for different times==
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| − | 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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| − | <html> | + | |
| − | <figure style="text-align:center;"> | + | |
| − | <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/10-4.png" alt="control">
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| − | <figcaption><b>Figure 10:</b>The RKO cell activity after incubation of plldR and pCadC control effector strains for different times under 3 day DMEM medium, normoxic conditions..</figcaption>
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| − | </figure>
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| − | </html> | + | |
| − | ==Western blot==
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| − | 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.
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| − | <html>
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| − | <figure style="text-align:center;">
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| − | <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/12-1.png" alt="control">
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| − | <figcaption><b>Figure 11:</b>Western blot result of HlyE under different promoter control</figcaption>
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| − | </figure>
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| − | </html>
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We used three different promoters in combination with XOR gate-HlyE to characterize the therapeutic efficacy of bacteria under the control of different promoters and TP901 coupled logic gates, and with the addition of lysis genes.
