Part:BBa_K2992048

FAST reporter construct with Pj23106 and thl RBS

Usage and Biology

This parts entry represents a FAST reporter construct under the regulatory control of Pj23106 from E. coli. The construct comprises the promoter Pj23106 BBa_J23106 coupled with the RBS from the C. acetobutylicum thiolase gene BBa_K2715009, driving the expression of the Fluorescence-Activating and Absorption-Shifting Tag protein (FAST) reporter gene BBa_K2992000. Transcriptional terminator occurs through the activity of the strong clostridial terminator TFad BBa_K2284012. FAST is one of the few fluorescent reporters available for effective use in anaerobic organisms. FAST is derived from Halorhodospira halophila and has been codon optimised for fluorescence studies in the genus Clostridium. In our project we couple FAST with the natural promoters of the BotR regulon thus linking reporter fluorescence with botulinum neurotoxin production. In doing so, we hoped to generate our surrogate host strain as a model for predicting neurotoxin production in foodstuffs following food manufacturing processes.

Characterisation

Data incoming.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 231
  

References

Streett, H., Kalis, K. and Papoutsakis, E. (2019). A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST). Applied and Environmental Microbiology, 85(14).

Heap, J., Pennington, O., Cartman, S. and Minton, N. (2009). A modular system for Clostridium shuttle plasmids. Journal of Microbiological Methods, 78(1), pp.79-85.