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| | pLldR-Bxb1 consists of a fusion of the lactate-sensitive promoter pLldR and the serine integrase Bxb1. It will act as a complex regulatory for controlling downstream logic gates and transcription of genes. | | pLldR-Bxb1 consists of a fusion of the lactate-sensitive promoter pLldR and the serine integrase Bxb1. It will act as a complex regulatory for controlling downstream logic gates and transcription of genes. |
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| − | ===Characterization===
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| − | ==In vitro characterization and data analysis of the reported strains==
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| − | To improve signaling stability as well as accuracy, we added Amplifying genetic switches based on serine integrase (Bxb1,TP901) to the R reporter(BBa_ ) to construct the AR reporter. Fig 1 indicates lactate (plldR) induced AR reporter with homogenized fluorescence intensity (mRFP/Cell). Comparing Fig1, 2, it can be seen that the fluorescence intensity of the AR reporter decreased significantly at a lactate concentration of 0 mM, and its expression was more stable over time. The fluorescence intensity of the AR reporter was also greater at other concentrations of lactate induction, and the difference between the fluorescence intensity after lactate induction at each concentration was more pronounced. This result indicates that the addition of amplifying genetic switch enhances the reporter intensity and robustness of the lactate biosensor.
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| − | <figcaption><b>Figure 1:</b> Induction of downstream gene mRFP expression over time by the AR reporter consisting of plldR+Switch +mRFP at different lactate concentrations.</figcaption>
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| − | <figcaption><b>Figure2:</b>Induction of downstream gene mRFP expression over time by the AR reporter consisting of plldR+mRFP at different lactate concentrations.</figcaption>
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| − | ==Lactate (plldR) and pH (pPepT)Induced promoter-controlled effector engineered strain co-incubated with RKO cells==
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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.
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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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| − | <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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| − | ==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.
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| − | <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.
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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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| | <span class='h3bb'>Sequence and Features</span> | | <span class='h3bb'>Sequence and Features</span> |
pLldR-Bxb1 is constructed with L-lactate-sensitive promoter pLldR and serine integrase Bxb1, and is uesd for lactate-specific response for logic gate coupling.
pLldR-Bxb1 consists of a fusion of the lactate-sensitive promoter pLldR and the serine integrase Bxb1. It will act as a complex regulatory for controlling downstream logic gates and transcription of genes.
