Difference between revisions of "Part:BBa K2992015"

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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 P<i>ntnH</i> ([https://parts.igem.org/Part:BBa_K2992001 BBa_K2992001]) in <i>C. botulinum</i>. ntnH encodes the non-toxic non-haemagglutinin component of the botulinum neurotoxin complexes. Its expression is directly regulated by BotR ([https://parts.igem.org/Part:BBa_K2992002 BBa_K2992002]).  In our project we use the regulatory region of <i>ntnH</i> to drive expression of the toxin regulator botR 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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The native 5’-UTR containing the RBS is found naturally upstream of P<i>ntnH</i> ([https://parts.igem.org/Part:BBa_K2992001 BBa_K2992001]) in <i>C. botulinum</i>. ntnH encodes the non-toxic non-haemagglutinin component of the botulinum neurotoxin complexes. Its expression is directly regulated by BotR ([https://parts.igem.org/Part:BBa_K2992002 BBa_K2992002]).  In our project we use the regulatory region of <i>ntnH</i> 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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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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<br>
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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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===References===
 
===References===
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Raffestin et al., 2005 (update)
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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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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2992015 parameters</partinfo>
 
<partinfo>BBa_K2992015 parameters</partinfo>
 
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Latest revision as of 02:54, 22 October 2019


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

5’-UTR containing RBS for ntnH gene from C. botulinum


Usage and Biology

The native 5’-UTR containing the RBS is found naturally upstream of PntnH (BBa_K2992001) in C. botulinum. ntnH encodes the non-toxic non-haemagglutinin component of the botulinum neurotoxin complexes. Its expression is directly regulated by BotR (BBa_K2992002). In our project we use the regulatory region of ntnH 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 using 3 difference reporters: GusA (BBa_K2992039), FAST (BBa_K2992044) and acetone (BBa_K2992036 ). This part was used as a promoter which functions in both the Gram-negative E. coli and the Gram-positive Clostridium sporogenens. 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.