Difference between revisions of "Part:BBa K2992049"

Latest revision as of 13:41, 21 October 2019

FAST reporter construct with PLac

Usage and Biology

This parts entry represents a FAST reporter construct under the regulatory control of the lactose-inducible PLac system from C. perfringens. The construct comprises the FAST reporter gene BBa_K2992000under the regulatory control of the BgaRL lactose inducible system from C. perfringens wherein the divergent PbgaRBBa_K2992020 and PbgaL BBa_K2992023 promoter sequences, coupled with their native 5’-UTR and RBS sequences BBa_K2992022 BBa_K2992024 respectively control the transcription of bgaR and FAST. Transcription for bgaR is terminated by its native terminator sequence BBa_K2992021

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.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 573
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 573
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 573
Illegal BglII site found at 633
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 573
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 573
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1600

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.

@@ Line 7: / Line 7: @@
 ===Usage and Biology===
-This parts entry represents a FAST reporter construct under the regulatory control of the lactose-inducible P<i>Lac</i> system from <i>C. perfringens</i>. The construct comprises the FAST reporter gene [https://parts.igem.org/Part:BBa_K2992000 BBa_K2992000]under the regulatory control of the BgaRL lactose inducible system from <i>C. perfringens</i> wherein the divergent P<i>bgaR</i>[https://parts.igem.org/Part:BBa_K2992020 BBa_K2992020] and P<i>bga</i>L [https://parts.igem.org/Part:BBa_K2992023 BBa_K2992023] promoter sequences, coupled with their native 5’-UTR and RBS sequences [https://parts.igem.org/Part:BBa_K2992022 BBa_K2992022][ https://parts.igem.org/Part:BBa_K2992024 BBa_K2992024] respectively control the transcription of <i>bgaR</i>  and <i>FAST</i>. Transcription for <i>bgaR</i> is terminated by its native terminator sequence [https://parts.igem.org/Part:BBa_K2992021 BBa_K2992021]
+This parts entry represents a FAST reporter construct under the regulatory control of the lactose-inducible P<i>Lac</i> system from <i>C. perfringens</i>. The construct comprises the FAST reporter gene [https://parts.igem.org/Part:BBa_K2992000 BBa_K2992000]under the regulatory control of the BgaRL lactose inducible system from <i>C. perfringens</i> wherein the divergent P<i>bgaR</i>[https://parts.igem.org/Part:BBa_K2992020 BBa_K2992020] and P<i>bga</i>L [https://parts.igem.org/Part:BBa_K2992023 BBa_K2992023] promoter sequences, coupled with their native 5’-UTR and RBS sequences [https://parts.igem.org/Part:BBa_K2992022 BBa_K2992022][https://parts.igem.org/Part:BBa_K2992024 BBa_K2992024] respectively control the transcription of <i>bgaR</i>  and <i>FAST</i>. Transcription for <i>bgaR</i> is terminated by its native terminator sequence [https://parts.igem.org/Part:BBa_K2992021 BBa_K2992021]
-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.   <br><br>
+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>
 <!-- -->
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K2992049 SequenceAndFeatures</partinfo>
+===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.
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