Difference between revisions of "Part:BBa K4156076"

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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 1 and 2, 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.

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

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 1). 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.

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