Part:BBa_K4156078
-- No description --
pPepT is a hypoxia-sensing promoter, designed to sense oxygen.
Usage and Biology
It is mainly regulated by the transcriptional activator (FNR). In the absence of oxygen, the FNR binds to the [4Fe-4S]2+ cluster to generate a transcriptionally active homodimer.[1] However, in the presence of oxygen, the [4Fe-4S]2+ cluster is degraded and the FNR dimer dissociates into inactive monomers.[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 hypoxia Promoter
To Characterize part，we first added mRFP after the promoter and wanted to initially test the response of this promoter to anoxia 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_K4156110
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_K4156108
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_K4156109
Better Chassis
Finally, based on the above validation, we can assume that strains were constructed that can stably respond to anoxia. 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 anoxia condition 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.
Applications
1.Western blot
To verify the extracellular secretion of HlyE, We connect pPepT 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 Yu B, Yang M, Shi L, et al. Explicit hypoxia targeting with tumor suppression by creating an "obligate" anaerobic Salmonella Typhimurium strain. Sci Rep. 2012;2:436. doi:10.1038/srep00436
2 Goers L, Ainsworth C, Goey CH, Kontoravdi C, Freemont PS, Polizzi KM. Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production. Biotechnol Bioeng. 2017;114(6):1290-1300. doi:10.1002/bit.26254
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]
- 1000COMPATIBLE WITH RFC[1000]
| None |