Part:BBa_K4156076
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
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
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
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 358
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