Part:BBa_K4156108

pPepT-TP901

pPepT-TP901 is constructed with hypoxia-sensing promoter pPepT and serine integrase TP901.

Usage and Biology

pPepT-TP901 consists of a fusion of the pH-sensitive promoter pPepT ( BBa_K4156078 ) and the serine integrase TP901 ( BBa_K4156087 ). It will act as a complex regulatory for controlling downstream logic gates and transcription of genes.

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_K4156118 ) to construct the AR reporter. Fig 1 indicates hypoxia (pPepT）induced AR reporters with homogenized fluorescence intensity (mRFP/Cell). Comparing Fig.1 and 2, the fluorescence expression of the AR reporter was significantly higher after the addition of Switch under anaerobic conditions. This result indicates that the addition of amplifying genetic switch enhances the reporter intensity and robustness of the lactate biosensor.

Figure 1: Induction of downstream gene mRFP expression over time by the AR reporter consisting of pPepT+Switch+mRFP under hypoxic and normoxic conditions.

Figure 2: Induction of downstream gene mRFP expression over time by the AR reporter consisting of pPepT +mRFP under hypoxic and normoxic conditions.

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.

Figure3:Fluorescence intensity of engineered bacterias with pLldR/pCadC/pPepT-Switch (TP901)-mRFP , versus control EcN 1917 ,after 48h of induction.

Hypoxia-inducible promoter (pPepT) control effector engineered strains were co-incubated with RKO cells.

We linked pPepT-TP901 to XOR gate-HlyE （ BBa_K4156119 ) for validation of treatment viability.

Figure 4 shows the RKO cell activity after incubation with each strain ((OD=0.6, 30 μl, 3 hours) in fresh DMEM medium, normoxic conditions. It can be seen that pPepT was not activated under normoxic conditions and the therapeutic protein was not expressed, so there was no significant change in RKO cell activity compared with the WT group. Figure5 shows the RKO cell activity of each strain after incubation in fresh DMEM medium, anoxic conditions. Under hypoxic conditions pPepT was activated and the expression of therapeutic proteins caused RKO cells to be killed, so RKO cell activity was significantly reduced compared to the WT group.

Figure 4:The activity of RKO cells after incubation with each strain (OD=0.6, 30 μl, 3 hours) in fresh DMEM medium, anoxic conditions

Figure 5:The activity of RKO cells after incubation with each strain (OD=0.6, 30 μl, 3 hours) in 3 day DMEM medium,anoxic conditions

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

We linked pPepT-TP901 to XOR gate-HlyE （ BBa_K4156119 ) for validation of treatment viability.

Figure 6 shows the RKO cell activity after incubation with different doses of pPepT-controlled effector strains under fresh DMEM medium, anoxic conditions. It can be seen that RKO cell activity decreases with increasing doses of hypoxia-inducible promoter-controlled effector strains.

Figure 6:The RKO cell activity after incubation with different doses of pPepT-controlled effector strains under fresh DMEM medium, anoxic conditions.

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

We linked pPepT-TP901 to XOR gate-HlyE （ BBa_K4156119 ) for validation of treatment viability.

From the figure 7, it can be obtained that RKO cell activity decreased with increasing incubation time of the effector strains controlled by pPepT under fresh DMEM medium, anoxic conditions.

Figure 7:The RKO cell activity decreased with increasing incubation time of the effector strains controlled by pPepT under fresh DMEM medium, anoxic conditions

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.

Figure 8:Western blot result of HlyE under different promoter control

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 1624
  
• 1000
  COMPATIBLE WITH RFC[1000]