Difference between revisions of "Part:BBa K2992015"
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| − | <partinfo> | + | <partinfo>BBa_K2992015 short</partinfo> |
| − | 5’-UTR containing RBS for <i>ntnH</i> gene from <i>C. botulinum</i> | + | 5’-UTR containing RBS for <i>ntnH</i> gene from <i>C. botulinum</i> |
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | The native 5’-UTR containing the RBS is found naturally upstream of <i>ntnH</i> 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. In our project we use the regulatory region of <i>ntnH</i> to drive expression of | + | 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=== | ||
| − | + | 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> | |
| + | <br> | ||
| + | https://2019.igem.org/wiki/images/5/5e/T--Nottingham--Basic4.png | ||
| + | <br> | ||
| + | https://2019.igem.org/wiki/images/6/6b/T--Nottingham--Basic3.png | ||
| + | <br> | ||
| + | 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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| + | <br> <br> <br> | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
| − | <partinfo> | + | <partinfo>BBa_K2992015 SequenceAndFeatures</partinfo> |
===References=== | ===References=== | ||
| − | Raffestin | + | 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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<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional 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.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.