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===Characterization===
===Characterization===
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==Lactate (plldR) and pH (pPepT)Induced promoter-controlled effector engineered strain co-incubated with RKO cells==
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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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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.
<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>
<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>
==Coincubation of different doses of effector engineered strains (OD=0.6) with RKO cells==
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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.
<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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==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.
<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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Latest revision as of 01:59, 12 October 2022
XOR gate-HlyE
XOR gate-HlyE consists of an XOR logic gate ( BBa_K4156117 ) and a downstream HlyE ( BBa_K4156084 ) sequence, and tests the performance of the XOR gate by detecting the red fluorescent signal.
Usage and Biology
The construction of this part is similar to XOR gate-mRFP ( BBa_K4156124 ). The difference is that we replaced the mRFP protein with the HlyE therapeutic protein. This is to verify the therapeutic power of the recombinant strain under XOR gate control. After co-incubation of the recombinant strain with cancer cells, cell viability was monitored using the CCK8 assay to assess the therapeutic power of the recombinant strain and the reinforcing effect of the XOR gate.
Characterization
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.
