Difference between revisions of "Part:BBa K2992014"

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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. This part was used as a promoter which functions in both the Gram-negative E. coli and the Gram-positive Clostridium <i>sporogenens</i>. 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 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
 
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 mild promoter in E.<i>coli</i> and only slightly stronger than no promoter in C.<i>sporogenes</i>. <br> In the C. <i>sporogenes</i> 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. <i>botulinum</i> promoters generated much lower levels of activity. Reporter activity appeared to be generally higher when analysed from the E. <i>coli</i> lysates as opposed to the C. <i>sporogenes</i> lysates. In those experiments, activity from the P<i>botR</i> and P<i>ntnh</i> constructs were considerably greater than the no promoter control. <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 E.coli and C.sporogenes.
  
 
[[File:Acetone data.png]]
 
[[File:Acetone data.png]]

Revision as of 02:10, 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 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 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.