Difference between revisions of "Part:BBa K2992001"

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===Usage and Biology===
 
===Usage and Biology===
  
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P<i>ntnh</i> is found upstream of the 5'-UTR and RBS of the non-toxic non-haemagglutinin component (ntnh) of the neurotoxin gene cluster in <i>C. botulinum</i>. This promoter was used to drive expression of of the toxin regulator <i>botR</i> and our chosen reporter genes of interest which have been placed under the control of the neurotoxin and neurotoxin-associated promoters. Doing so allows us to link volatile reporter production with neurotoxin production following food manufacturing processes.
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P<i>ntnh</i> is found upstream of the 5'-UTR and RBS of the non-toxic non-haemagglutinin component (ntnh) of the neurotoxin gene cluster in <i>C. botulinum</i>. This promoter was used to drive expression of our chosen reporter genes of interest. Doing so, allowed us to link BotR expression with the transcriptional activation of our reporter genes.
  
 
===Characterisation===
 
===Characterisation===
  
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This basic part was used for the assembly of our composite parts and characterised using using 3 difference reporters: [https://parts.igem.org/Part:BBa_K2992039 GusA (BBa_K2992039)], [https://parts.igem.org/Part:BBa_K2992044 FAST (BBa_K2992044)] and [https://parts.igem.org/Part:BBa_K2992036 acetone (BBa_K2992036 )]. 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 3 difference reporters: [https://parts.igem.org/Part:BBa_K2992039 GusA (BBa_K2992039)], [https://parts.igem.org/Part:BBa_K2992044 FAST (BBa_K2992044)] and [https://parts.igem.org/Part:BBa_K2992036 acetone (BBa_K2992036 )]. 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
 
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>botR</i> using FAST fluorescent assay, showed P<i>ntnh</i> to be an effective 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.
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Characterisation of this promoter (comprising parts [https://parts.igem.org/Part:BBa_K2992001 BBa_K2992001] and [https://parts.igem.org/Part:BBa_K2992015 BBa_K2992015]) against P<i>fdx</i>, P<i>thl</i> and P<i>botR</i> using FAST fluorescent assay, showed that P<i>ntnh</i> is a medium-strength promoter in E.<i>coli</i>, while being very weak in C.<i>sporogenes</i> (only slightly stronger than the promoterless control), as expected in the absence of BotR. <br>  
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Latest revision as of 02:55, 22 October 2019


PntnH

Promoter region for the non-toxic non-haemagglutinin component of the neurotoxin gene cluster in Clostridum botulinum.

Usage and Biology

Pntnh is found upstream of the 5'-UTR and RBS of the non-toxic non-haemagglutinin component (ntnh) of the neurotoxin gene cluster in C. botulinum. This promoter was used to drive expression of our chosen reporter genes of interest. Doing so, allowed us to link BotR expression with the transcriptional activation of our reporter genes.

Characterisation

This basic part was used for the assembly of our composite parts and characterised using 3 difference reporters: GusA (BBa_K2992039), FAST (BBa_K2992044) and acetone (BBa_K2992036 ). More information can be found on our Results Page.



T--Nottingham--Basic4.png
T--Nottingham--Basic3.png
Characterisation of this promoter (comprising parts BBa_K2992001 and BBa_K2992015) against Pfdx, Pthl and PbotR using FAST fluorescent assay, showed that Pntnh is a medium-strength promoter in E.coli, while being very weak in C.sporogenes (only slightly stronger than the promoterless control), as expected in the absence of BotR.


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.