Part:BBa_K2244009

ColE promoter+mCherry gene+T1 terminator+Constitutive promoter+Lev1 gene+T1terminator

This part is a functional composite part/device of a light-repressed expression system that is once activated will transcribe the reporter gene mCherry (BBa_K2244008). This device is named the lightOFF system.

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.

-mCherry (BBa_K2244008) is a red fluorescent protein used as a reporter gene. It is based on a fluorescent protein originally isolated from Discosoma sp. In this project, mCherry sequence was codon optimized for E. coli system.

-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 to express mCherry fluoresence protein under the control of light (Figure). By replacing mCherry with pesticide degrading genes (mheI, BBa_K2244004) or (TorA-opdA, BBa_K2244003) to produce pesticide degrading hydrolase in a light-regulated manner.

Figure: Cell growth and mCherry expression study in lightOFF system. Wildtype DH5a cells transformed with pLEV1(408) vector. The cells were illuminated with blue light or wrapped in aluminium foil to induce mCherry expression. Fluorescence and OD600 were measured over a 20-h period of time. a.u. arbitary units

Reference

1)Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, et al. (2005) Synthetic biology: Engineering Escherichia coli to see light. Nature 438: 441–442.

2)Tabor, J. J., Levskaya, A. & Voigt, C. A, 2011. Multi-chromatic Control of Gene Expression in Escherichia coli. J. Mol.Biol. 405:315–324.

3)Chen, X., Liu, R., Ma, Z., Xu, X, Zhang, H., Xu, J. & Yang, 2016. An extraordinary stringent and sensitive light-switchable gene expression system for bacterial cells. Cell Research, 26 (7): 854-7.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1015
  Illegal NheI site found at 1038
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 845
  Illegal AgeI site found at 1753
  
• 1000
  COMPATIBLE WITH RFC[1000]