Difference between revisions of "Part:BBa K2992012"

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===Usage and Biology===
 
===Usage and Biology===
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This promoter region is found naturally upstream of the <i>botR</i>  5’-UTR in <i>C. botulinum</i> [https://parts.igem.org/Part:BBa_K2992014 BBa_K2992014]. BotR is an alternative sigma factor involved in the positive regulation of botulinum neurotoxin and associated genes. In our project, we use P<i>botR</i> to drive the expression of <i>botR</i>  ([https://parts.igem.org/Part:BBa_K2992002 BBa_K2992002]) which in turn, regulates the production of our volatile reporter genes which we have placed under the control of neurotoxin and neurotoxin-associated promoters ([https://parts.igem.org/Part:BBa_K2992028 BBa_K2992028], [https://parts.igem.org/Part:BBa_K2992029 BBa_K2992029], [https://parts.igem.org/Part:BBa_K2992030 BBa_K2992030], [https://parts.igem.org/Part:BBa_K2992034 BBa_K2992034], [https://parts.igem.org/Part:BBa_K2992035 BBa_K2992035], [https://parts.igem.org/Part:BBa_K2992036 BBa_K2992036]). Doing so allows us to link volatile reporter production with neurotoxin production following food manufacturing processes.      <br><br>
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This promoter region is found naturally upstream of the <i>botR</i>  5’-UTR in <i>C. botulinum</i> [https://parts.igem.org/Part:BBa_K2992014 BBa_K2992014]. BotR is an alternative sigma factor involved in the positive regulation of botulinum neurotoxin and associated genes. In our project, we use P<i>botR</i> to drive the expression of <i>botR</i>  ([https://parts.igem.org/Part:BBa_K2992002 BBa_K2992002]) which in turn, regulates the production of our reporter genes which we have placed under the control of a BotR-activated promoter ([https://parts.igem.org/Part:BBa_K2992028 BBa_K2992028], [https://parts.igem.org/Part:BBa_K2992029 BBa_K2992029], [https://parts.igem.org/Part:BBa_K2992030 BBa_K2992030], [https://parts.igem.org/Part:BBa_K2992034 BBa_K2992034], [https://parts.igem.org/Part:BBa_K2992035 BBa_K2992035], [https://parts.igem.org/Part:BBa_K2992036 BBa_K2992036]). Doing so allows us to link reporter production with the presence of BotR.      <br><br>
  
 
===Characterisation===
 
===Characterisation===

Revision as of 01:04, 22 October 2019


PbotR from C. botulinum

Promoter region for botR in C. botulinum


Usage and Biology

This promoter region is found naturally upstream of the botR 5’-UTR in C. botulinum BBa_K2992014. BotR is an alternative sigma factor involved in the positive regulation of botulinum neurotoxin and associated genes. In our project, we use PbotR to drive the expression of botR (BBa_K2992002) which in turn, regulates the production of our reporter genes which we have placed under the control of a BotR-activated promoter (BBa_K2992028, BBa_K2992029, BBa_K2992030, BBa_K2992034, BBa_K2992035, BBa_K2992036). Doing so allows us to link reporter production with the presence of BotR.

Characterisation

This basic part was used for the assembly of our composite parts and characterised using using FAST and acetone assays. This part was used as a promoter which functions in both the Gram-negative E. coli and the Gram-positive Clostridium sporogenens. More information can be found on our Results Page.



T--Nottingham--Basic4.png
T--Nottingham--Basic3.png
Characterisation of this promoter against Pfdx, Pthl and Pntnh using FAST fluorescent assay, showed PBotR to be a mild promoter in E.coli and only slightly stronger than no promoter in C.sporogenes.
In the C. sporogenes experiments, adequate expression was detected for each of the clostridial promoters chosen for study. The two Pfdx derivatives generated the greatest level of reporter activity whilst the two C. botulinum promoters generated much lower levels of activity. Reporter activity appeared to be generally higher when analysed from the E. coli lysates as opposed to the C. sporogenes lysates. In those experiments, activity from the PbotR and Pntnh constructs were considerably greater than the no promoter control.

Acetone data.png
The data demonstrated appreciable acetone production of >2nM concentration when using either the native PbotR promoter and associated 5’-UTR+RBS or the RBS only construct to permit polar transcription from PpyrKDE. Considerable acetone production (4-6nM) was observed when using the constitutive clostridial promoter Pfdx. Crucially, acetone production was comparably scant when botR was absent from the genome of C. sporogenes and when no promoter was used to drive expression of the acetone production operon. These data provide experimental validation for the production of acetone in C. sporogenes as a model for Botulinum toxin prediction in foodstuffs.


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
    COMPATIBLE WITH RFC[1000]

References

Raffestin, S., Dupuy, B., Marvaud, J. and Popoff, M. (2004). BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani. Molecular Microbiology, 55(1), pp.235-249.

Zhang, Z., Korkeala, H., Dahlsten, E., Sahala, E., Heap, J., Minton, N. and Lindström, M. (2013). Two-Component Signal Transduction System CBO0787/CBO0786 Represses Transcription from Botulinum Neurotoxin Promoters in Clostridium botulinum ATCC 3502. PLoS Pathogens, 9(3), p.e1003252.