Difference between revisions of "Part:BBa K4156076"

Latest revision as of 07:33, 13 October 2022

Promoter pCadC

pCadC is a pH-sensitive promoter, designed to response the low-pH conditions.

Usage and Biology

pCadC is regulated by membrane-tethered activator protein (CadC), exhibits higher activity in acidic media than in media at neutral pH. In pH reporter strains, it’s used to test their response to acidic conditions in tumors induction.^[1-2]

Characterization

In order to verify the response sensitivity as well as the signal output effect of this new promoter, four iterations of experiments were conducted. The specific characterization is as follows.

Initial Testing of pH Promoter

To Characterize part，we first added mRFP after the promoter and wanted to initially test the response of this promoter to low pH based on the fluorescence intensity. E. coli Nissle 1917 was used as chassis.Details of the characterization and test results can be found at BBa_K4156111

We constructed a pH reporter consisting of the pH-inducible promoter pCadC+mRFP. To test itsperformance, we added reporter in different chassis organisms. Fig 1 illustrates that pCadC induces the expression of the downstream gene mRFP with the decrease of pH,. Thus, it can be seen that pH reporter can work properly.

Stability improvement

Then，amplifying genetic switches and Boolean logic gates based on serine integrase (TP901) are used in the design of biosensor systems ^[3]. These genetic devices enable bacteria to perform reliable detection, multiplex logic and data storage of clinical biomarkers in human clinical samples ^[4-5] to meet the requirements of medical testing. For characterization, we added switch, which is TP901 and XOR gate, then followed with mRFP. Details of the characterization and test results can be found at BBa_K4156099

1.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 pH (pCadc) induced AR reporters with homogenized fluorescence intensity (mRFP/Cell). In contrast to Fig 2 and 3, the fluorescence intensity of the AR reporter appeared more stable over time at pH 7.3 and was higher than that of the R reporter at pH 5.8, 6.3, and 7.3. 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 4), indicating that the pLldR/pCadC/PepT-Switch (TP901) system could be expressed normally.

2.Engineered strain co-incubated with RKO cells

Details of this section can be found in the next column "Addition of lysis genes"

Addition of lysis genes

Because we have therapeutic proteins that cannot be exocytosed, it is not enough to simply stabilize the response signal, and we intend to add bacteriophage lysis gene phiX174E parts that will enable bacteria lysis.So next we added phiX174E to the above genetic parts. Details of the characterization and test results can be found at BBa_K4156100

1. In vitro characterization and data analysis of the reported strains with φ174E

We constructed the lysis reporter CR by adding pH-sensing promoter followed by the amplification genes Switch and mRFP.( BBa_K4156118 )

Fig5 indicates pH (pCadc) inducing reporters after the addition of the lysis gene φ174E in induced and non-induced .The lower OD600 values indicate better lysis of the bacteria. Fig1, as the pH decreases, the OD600 value also decreases,indicating that our constructed strain can respond well to the tumor environment.

Fig6 indicates the fluorescence intensity of pH (pCadc) induced reporters under induced and non-induced conditions after the addition of lysis geneφ174E.The fluorescence intensity showed an upward trend with decreasing pH, and the fluorescence intensity under normoxic conditions was very low, while the fluorescence intensity under hypoxic conditions increased significantly after 8h.

Fig7 is the OD600 of wild-type 1917 bacteria under induced and non-induced conditions, and the wild-type bacteria could hardly respond to the induction of pH environments. The results show that CR undergoes lysis under induced conditions, but the cells still produce fluorescence. It indicates that the fitted set of equations for lysis-growth should be a resonance function.

To further obtain the lysis-growth curve, we shortened the assay time to 5 min a measurement . Fig8, OD600 changes of pH(pCadc)-induced reporter under induced and non-induced conditions.The results indicate that the lysis-growth curve is a dynamic function.

Next, we tested the constructed CR reporters using CT26 cell cultures. In Fig9,10, CT26 cells were cultured for 5 consecutive days, and the OD600 values and fluorescence response of the pCadC-controlled CR were tested by measuring the pH after collecting the cell supernatant every 12 hours and using this sample as the medium; In Fig 5 and 6, the pH level of the above cell culture medium sample was measured and used as the medium to test the OD600 value and fluorescence response of the pCadC-controlled CR. Fig5, As the pH decreased, more bacteria were lysed and the OD600 values showed a decreasing trend. Fig6, the fluorescence intensity shows an increasing trend as the pH decreases. The results indicate that CR reporters can respond in cell culture medium.

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

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

Figure 11 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 12 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 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 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.

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

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

Figure 13 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.

4. 30 μl effector engineered strains (OD=0.6) were co-incubated with RKO cells for different times

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

Figure 14 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.

Better Chassis

Finally, based on the above validation, we can assume that strains were constructed that can stably respond to low pH. Since the chassis organism must be E. coli, but we started to think in which strain this gene circuit is responding better. So we compared it in E. coli Nissle 1917 and E. coli DH 5-alpha. The data were recorded at 2-hour intervals over 48 hours of induction at the same four pH values as before, and finally plotted as the normalized fluorescence intensity (figure 15). It can be observed that the circuit responds with higher intensity in E. coli Nissle 1917 than in E. coli DH5-alpha, so E. coli Nissle 1917 is a better chassis organism.

Applications

1.Western blot

To verify the extracellular secretion of HlyE, We connect pCadC to HlyE and 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.

References

1 Schlundt A, Buchner S, Janowski R, et al. Structure-function analysis of the DNA-binding domain of a transmembrane transcriptional activator. Sci Rep. Apr 21 2017;7(1):1051. doi:10.1038/s41598-017-01031-9

2 Lee YH, Kim JH, Bang IS, Park YK. The membrane-bound transcriptional regulator CadC is activated by proteolytic cleavage in response to acid stress. J Bacteriol. Jul 2008;190(14):5120-6. doi:10.1128/jb.00012-08

3 Courbet A, Endy D, Renard E, Molina F, Bonnet J. Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates. Sci Transl Med. May 27 2015;7(289):289ra83. doi:10.1126/scitranslmed.aaa3601

4 Benenson Y. Biomolecular computing systems: principles, progress and potential. Nat Rev Genet. Jun 12 2012;13(7):455-68. doi:10.1038/nrg3197

5 Bonnet J, Yin P, Ortiz ME, Subsoontorn P, Endy D. Amplifying genetic logic gates. Science. May 3 2013;340(6132):599-603. doi:10.1126/science.1232758

Sequence and Features