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>. 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 (add hyperlinks). 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 allowed us to link BotR expression with the trasncriptional activation of our reporter genes.      <br><br>
  
 
===Characterisation===
 
===Characterisation===
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Data incoming
 
  
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This basic part was used for the assembly of our composite parts and was characterised using FAST, GusA and acetone assays. More information can be found on our [https://2019.igem.org/Team:Nottingham/Results Results Page].<br> <br> <br>
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<span class='h3bb'>Sequence and Features</span>
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<br>
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<partinfo>BBa_K2992012 SequenceAndFeatures</partinfo>
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https://2019.igem.org/wiki/images/5/5e/T--Nottingham--Basic4.png
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https://2019.igem.org/wiki/images/6/6b/T--Nottingham--Basic3.png
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Characterisation of P<i>botR</i> (comprising parts [https://parts.igem.org/Part:BBa_K2992012 BBa_K2992012] and [https://parts.igem.org/Part:BBa_K2992014 BBa_K2992014]) against Pfdx, Pthl and Pntnh using the FAST fluorescent assay, showed PbotR to be a relatively weak promoter in both <i>E.coli</i> and <i>C.sporogenes</i>.
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<partinfo>BBa_K2992000 SequenceAndFeatures</partinfo>
  
 
===References===  
 
===References===  
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Raffestin et al., 2009 to be updated.
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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.  
  
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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.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 02:48, 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 allowed us to link BotR expression with the trasncriptional activation of our reporter genes.

Characterisation

This basic part was used for the assembly of our composite parts and was characterised using FAST, GusA and acetone assays. More information can be found on our Results Page.



T--Nottingham--Basic4.png
T--Nottingham--Basic3.png
Characterisation of PbotR (comprising parts BBa_K2992012 and BBa_K2992014) against Pfdx, Pthl and Pntnh using the FAST fluorescent assay, showed PbotR to be a relatively weak promoter in both E.coli and C.sporogenes.


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 155

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.