Difference between revisions of "Part:BBa K2992047"
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===Usage and Biology===
 
===Usage and Biology===
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This parts entry represents a FAST reporter construct under the regulatory control of P<i>ha33</i> for the hemagglutinin component of the Botulinum neurotoxin complex in <i>C. botulinum</i>. The construct comprises the promoter P<i>ha33</i> [https://parts.igem.org/Part:BBa_K2992040 BBa_K2992040] coupled with its associated 5’-UTR containing the RBS [https://parts.igem.org/Part:BBa_ K272992041 BBa_ K2992041], driving the expression of the Fluorescence-Activating and Absorption-Shifting Tag protein (FAST) reporter gene [http//parts.igem.org/Part:BBa_K2992000 BBa_K2992000]. Transcirptional terminator occurs through the activity of the strong clostridial terminator T<i>Fad</i> [https://parts.igem.org/Part:BBa_K2284012 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 <i>Clostridium</i>. 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.  <br><br>
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This parts entry represents a FAST reporter construct under the regulatory control of P<i>ha33</i> for the hemagglutinin component of the Botulinum neurotoxin complex in <i>C. botulinum</i>. The construct comprises the promoter P<i>ha33</i> [https://parts.igem.org/Part:BBa_K2992040 BBa_K2992040] coupled with its associated 5’-UTR containing the RBS [https://parts.igem.org/Part:BBa_K272992041 BBa_K2992041], driving the expression of the Fluorescence-Activating and Absorption-Shifting Tag protein (FAST) reporter gene [https://parts.igem.org/Part:BBa_K2992000 BBa_K2992000]. Transcriptional terminator occurs through the activity of the strong clostridial terminator T<i>Fad</i> [https://parts.igem.org/Part:BBa_K2284012 BBa_K2284012]. FAST is one of the few fluorescent reporters available for effective use in anaerobic organisms. FAST is derived from <i>Halorhodospira halophila</i> and has been codon optimised for fluorescence studies in the genus <i>Clostridium</i>. 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.  <br><br>
 
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===References===
 
===References===
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Heap modular  
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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).
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Street et al 2019. Update
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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.
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Latest revision as of 13:43, 21 October 2019


FAST reporter construct with Pha33 5-UTR+RBS


Usage and Biology

This parts entry represents a FAST reporter construct under the regulatory control of Pha33 for the hemagglutinin component of the Botulinum neurotoxin complex in C. botulinum. The construct comprises the promoter Pha33 BBa_K2992040 coupled with its associated 5’-UTR containing the RBS BBa_K2992041, 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
    COMPATIBLE WITH RFC[12]
  • 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 276

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.