Part:BBa_K2244010

ColE promoter +supernova gene +Lev1 gene

The device is a functional composite part containing a suicide gene supernova (BBa_K1491017).

Biology

-ColE 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) by replacing mCherry gene with supernova. This is to allow supernova to be firstly induced in darkness and then secrete ROS upon light illumination which promotes cell death (Figure 1-2). This allows a good incorperation of suicide system into our light-regulated expression system.

Figure 1: 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.

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Figure 2: 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 during a 48-h period of time. LB plates were grown for 24 h after light irradation/kept in darkenss.

Improvement

This part is an improved part of supernova (BBa_K1491017). The improvement was made by inserting supernova gene into a light-regulated expression system, lightoff (BBa_K2244009), cell apoptosis can thus be induced by firstly expressing supernova gene in darkness, and then expose cells to light illumination that results to ROS release and the subsequent cell death. The entire process is purely regulated by light. We believe this has improved the original use of supernova in a T7 system using IPTG induction, which requires additional chemical inducer.This improvement has made the use of supernova suicide system more convenient and more easily manipulated.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1045
  Illegal NheI site found at 1068
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 213
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1783
  
• 1000
  COMPATIBLE WITH RFC[1000]