Difference between revisions of "Part:BBa K2992014"

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===Usage and Biology===
 
===Usage and Biology===
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The native 5’-UTR containing the RBS is found naturally upstream of <i>botR</i> 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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The native 5’-UTR containing the RBS is found naturally upstream of <i>botR</i> 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 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 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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https://2019.igem.org/wiki/images/5/5e/T--Nottingham--Basic4.png
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<br>
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https://2019.igem.org/wiki/images/6/6b/T--Nottingham--Basic3.png
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<br>
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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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[[File:Acetone data.png]]
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<br>
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The data demonstrated appreciable acetone production of >2nM concentration when using either the native P<i>botR</i> promoter and associated 5’-UTR+RBS or the RBS only construct to permit polar transcription from P<i>pyrKDE</i>. Considerable acetone production (4-6nM) was observed when using the constitutive clostridial promoter P<i>fdx</i>. Crucially, acetone production was comparably scant when <i>botR</i> was absent from the genome of <i>C. sporogenes</i> 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 <i>C. sporogenes</i> as a model for Botulinum toxin prediction in foodstuffs.
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===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.
  
  

Latest revision as of 02:44, 22 October 2019


5-UTR containing RBS for botR gene from C. botulinum

5’-UTR containing RBS region for botR gene in C. botulinum


Usage and Biology

The native 5’-UTR containing the RBS is found naturally upstream of botR in C. botulinum. 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 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.

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.