Part:BBa_K2244010
ColE promoter +supernova gene +Lev1 gene
The device is a functional composite part containing a suicide gene supernova (BBa_K1491017).
Biology
-Co1E promoter (BBa_K2244006) is derived from the promoter region of colicin E gene located in the ColE1 plasmid of E.coli. ColE promoter contains a ‘SOS’ operator region that allows the binding of LexA protein to repress transcription. DNA-binding component of LexA repressor in LEV1 would form a dimer and bind to the operator sequence thus halts the activity of ColE promoter.
-Supernova(BBa_K1491017) encodes a phototoxic compound, which is a mutant form of KillerRed(BBa_K1184000), the first genetically-encoded photosensitizer. Upon illumination, reactive oxygen species (ROS) are generated to induce cell apoptosis. KillerRed is engineered from anm2CP to be phototoxic.
-LEV1 repressor (BBa_K2244005) is a fusion protein of VVD and LexA. Blue light sensor VIVID was derived from the chromosome of Neurospora crassa. The LOV domain of the protein VVD has the capacity to self-dimerize upon light stimulation. LexA repressor is a transcriptional repressor of SOS regulon in E.coli. LEV1 is the core component of this device.
-Constitutive promoter (BBa_K2244012), in this device, it is used to constitutively express Lev1 gene.
-T1 terminator (BBa_B0010), it is the most used terminator in E. coli system
Usage
In our project this year, this device worked in the lightOFF system (BBa_K2244009) to replace the ColE promoter+mCherry+T1 terminator section and to allow the supernova to be induced in darkness and start secreting ROS upon light irradiation that promotes cell death.
Figure: the apoptosis effect of supernova to E. coli cells. Light exposure greatly reduced the CFU number of bacteria cells, while darkness exposure allowed normal growth. Inert: supernova expression indicated by pink cells.
Reference
1) Bulina, M. E., Chudakov, D. M., Britanova, O. V. & Lukyanov. K.. 2003. A genetically encoded photosensitizer. Nat. Biotechnol. 24, 95-99.
2)Tour, O., Meijer, R. M., Zacharias, D. A., Adams, S. R. & Tsien, R. Y, 2003. Genetically targeted chromophore-assisted light inactivation. Nat. Biotechnol. 21, 1505–1508.
3)Wong, E. V., David, S., Jacob, M. H. & Jay, D. G, 2003. Inactivation of myelin-associated glycoprotein enhances optic nerve regeneration. J. Neurosci. 23, 3112–3117.
4)Takemoto, K., Matsuda, T., Sakai, N., Fu, D., Noda, M., Uchiyama, S., Kotera, I., Arai, Y., Horiuchi, M., Fukui, K. and Ayabe, T., 2013. SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation. Scientific reports.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1045
Illegal NheI site found at 1068 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 213
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1783
- 1000COMPATIBLE WITH RFC[1000]
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