Difference between revisions of "Part:BBa K4156104"

Revision as of 07:23, 13 October 2022

pLldR-TP901

pLldR-TP901 is constructed with L-lactate-sensitive promoter pLldR and serine integrase TP901.

Usage and Biology

pLldR-TP901 consists of a fusion of the lactate-sensitive promoter pLldR ( BBa_K4156101 ) and the serine integrase TP901( BBa_K4156112 ). It will act as a complex regulatory for controlling downstream logic gates and transcription of genes.

Characterization

In vitro characterization and data analysis of the reported strains

To improve signaling stability as well as accuracy, we added Amplifying genetic switches based on serine integrase (TP901) to the R reporter( BBa_K4156115 ) 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.

We also observed the mRFP fluorescence intensity of WT 1917 and reporter strain AR(pLldR/pCadC/pPepT-Switch (TP901)-mRFP) after 48 h of induction using a fluorescence microscope (Olympus BX53). The results showed that the three promoters (pLldR, pCadC and pPepT)-Switch (TP901)-mRFP exhibited a uniform and clear red fluorescence signal after induction(Fig 3), indicating that the pLldR/pCadC/PepT-Switch (TP901) system could be expressed normally.

3.Lactate induced promoter-controlled effector engineered strain co-incubated with CT26 cells

For the pLldR-Switch(TP901)-HlyE transformed effector, it was co-incubated with CT26 cells and ECN 1917 was used as a control. CT26 cells after incubation with different bacteria were assayed using Calcein/PI Cell Activity and Cytotoxicity Assay Kit (C2015S, Biotime) with wild-type E. coli 1917 as control and observed using fluorescence microscopy. The principle of the kit is that two probes can detect intracellular esterase activity and cell membrane integrity, respectively, thus reflecting cell activity and cytotoxicity. Calcein AM stains live cells with green fluorescence, while Propidium Iodide (PI) stains dead cells with red fluorescence.

The results showed that the green fluorescence of cells stained by Calcein AM decreased significantly after co-incubation with the engineered bacteria, while the red fluorescence of cells stained by PI increased significantly(Fig 4). It was demonstrated that the cell activity was significantly reduced and the toxicity was significantly increased under the co-incubation of the engineered bacteria, i.e. the pLldR-Switch(TP901)-HlyE system had a significant effect on the treatment of tumor cells with CT26.

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

Figure 1 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 2 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 to XOR gate-HlyE （ BBa_K4156119 ) for validation of treatment viability.

Figure 1 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 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