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                 <figcaption><b>Figure 2:</b>  Induction of downstream gene mRFP expression over time by the AR reporter consisting of pPepT +mRFP under hypoxic and normoxic conditions.</figcaption>
 
                 <figcaption><b>Figure 2:</b>  Induction of downstream gene mRFP expression over time by the AR reporter consisting of pPepT +mRFP under hypoxic and normoxic conditions.</figcaption>
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==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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                <figcaption><b>Figure 3:</b>Western blot result of HlyE under different promoter control</figcaption>
 
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Revision as of 03:31, 11 October 2022


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 and the serine integrase TP901. 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 (Bxb1,TP901) to the R reporter(BBa_ ) 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.

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

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

control
Figure 3: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]