Difference between revisions of "Part:BBa K2992012"

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===Characterisation===
 
===Characterisation===
  
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This basic part was used for the assembly of our composite parts and characterised using using FAST 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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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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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 this promoter against P<i>fdx</i>, P<i>thl</i> and P<i>ntnh</i> using FAST fluorescent assay, showed P<i>botR</i> to be a relatively weak promoter in both E.<i>coli</i> and and C.<i>sporogenes</i>. <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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<partinfo>BBa_K2992000 SequenceAndFeatures</partinfo>
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The data demonstrated acetone production of >2mM when using P<i>botR</i> promoter or P<i>pyrKDE</i> to drive expression of <i>botR</i> in the chromosome of <i>C. sporogenes</i>. Considerable acetone production (4-6 mM) was observed when using the constitutive clostridial promoter P<i>fdx</i>, driving directly the expression of the acetone operon genes. Crucially, acetone production was almost undetectable 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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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2992012 SequenceAndFeatures</partinfo>
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===References===  
 
===References===  

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.