Part:BBa_K4375019
Blue light inducible production of Cytolysin A (BLADE)
This device is meant for the light-inducible production of Cytolysin A (BBa_K811000), a pore-forming toxin. To achieve this, we used a modified arabinose operator system, where the transcription factor, AraC, is engineered to homodimerize upon blue-light illumination instead of arabinose binding. This system is called BLADE, and it is produced under the strong constitutive promoter J23101*(BBa_K4375003).
Characteristics
We aimed to characterise the Cytolysin A production BLADE Expression System using SDS-PAGE, and study its hemolytic activity on blood agar plates. (Fig 1.) For the Cytolysine production characterisation, we obtained the supernatant of the bacterial culture after 4 h of blue light illumination and precipitated its protein content. We observed a band indicating Cytolysin A production, where we expected, which was absent in the case of the dark control sample. (Fig. 1/B) We implemented the protocol of Chiang et al. for this aim, but it should be noted, that this experiment needs to be repeated with a construct to which an affinity tag, such as 6xHis-Tag, is added. [3] In this way, the protein of our interest could be purified lessening the number of aspecific bands. We also meant to assess the hemolytic activity of Cytolysin A, using the BLADE Expression System. We spread the overnight cultures on Blood Agar plates, grew them for 8 h in dark at 37°C, which was followed by an 4 h long illumination with blue light at 25 °C. (The dark control plate was kept in dark at 25 °C for that time.) For additional negative control, we also spread BLADE-mCherry sample on the plates.) As we expected, no hemolytic activity was observed in the case of mCherry. Regarding the BLADE-ClyA samples, the biological activity of Cytolysine A was undoubtful since the blood agar discoloured where the samples were plated. Unfortunately, we could not observe a significant difference between dark and illuminated plates. This could also be explained by the considerations we beforehand mentioned. (Temperature, frozen bacterial culture)
Usage and Biology
This device is a blue light-activable system, which is based on the BLADE protein. Upon illumination, the BLADE binds to the AraBAD promoter and Cytolysin-A (ClyA) is expressed. ClyA is a protein native to E. coli and Salmonella typhi that is capable of forming pore complexes (BBa_K811000). ClyA is a 34 kDa protein and has alpha-helical-based monomers which oligomerize and form the pore. The picture shows the construction scheme
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 29 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
References
Romano, E.; Baumschlager, A.; Akmeriç, E. B.; Palanisamy, N.; Houmani, M.; Schmidt, G.; Öztürk, M. A.; Ernst, L.; Khammash, M.; Di Ventura, B. Engineering AraC to Make It Responsive to Light Instead of Arabinose. Nature Chemical Biology 2021, 17 (7), 817–827. https://doi.org/10.1038/s41589-021-00787-6.
Peraro, M. D.; van der Goot, F. G. Pore-Forming Toxins: Ancient, but Never Really out of Fashion. Nature Reviews Microbiology 2015, 14 (2), 77–92. https://doi.org/10.1038/nrmicro.2015.3
Sathyanarayana, P.; Maurya, S.; Behera, A.; Ravichandran, M.; Visweswariah, S. S.; Ayappa, K. G.; Roy, R. Cholesterol Promotes Cytolysin A Activity by Stabilizing the Intermediates during Pore Formation. Proceedings of the National Academy of Sciences 2018, 115 (31). https://doi.org/10.1073/pnas.1721228115.
Li, Y.; Li, Y.; Mengist, H. M.; Shi, C.; Zhang, C.; Wang, B.; Li, T.; Huang, Y.; Xu, Y.; Jin, T. Structural Basis of the Pore-Forming Toxin/Membrane Interaction. Toxins 2021, 13 (2), 128. https://doi.org/10.3390/toxins13020128.
Morton, C. J.; Sani, M.-A.; Parker, M. W.; Separovic, F. Cholesterol-Dependent Cytolysins: Membrane and Protein Structural Requirements for Pore Formation. Chemical Reviews 2019, 119 (13), 7721–7736. https://doi.org/10.1021/acs.chemrev.9b00090.
Murase, K. Cytolysin A (ClyA): A Bacterial Virulence Factor with Potential Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy. Toxins 2022, 14 (2), 78. https://doi.org/10.3390/toxins14020078.
Schwechheimer, C., Kuehn, M. Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nat Rev Microbiol 13, 605–619 (2015). https://doi.org/10.1038/nrmicro3525
Chiang, C.-J.; Huang, P.-H. Metabolic Engineering of Probiotic Escherichia Coli for Cytolytic Therapy of Tumors. Scientific Reports 2021, 11 (1). https://doi.org/10.1038/s41598-021-85372-6.
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