Difference between revisions of "Part:BBa K2992017"
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<partinfo>BBa_K2992017 short</partinfo>
 
<partinfo>BBa_K2992017 short</partinfo>
  
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Promoter region from the ferredoxin gene of <i>C. sporogenes</i> with the corrected t14c substitution compared with Part:BBa_K2715002 (add hyperlink).
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5’-UTR containing RBS for <i>fdx</i> gene from <i>C. sporogenes</i>
  
  
 
===Usage and Biology===
 
===Usage and Biology===
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Ferredoxin is an iron-sulfur protein involved in central redox reactions occurring during the general metabolism of Clostridia. 5’-UTR and RBS region for fdx is found naturally downstream of Pfdx and upstream of the start codon. In our project we use the regulatory region for the fdx gene comprising Pfdx-t14c (hyperlink) and this entry to regulate our volatile reporter genes for predicting the production of botulinum neurotoxin following food manufacture. <br><br>
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Ferredoxin is an iron-sulfur protein involved in central redox reactions occurring during the general metabolism of Clostridia. 5’-UTR and RBS region for fdx is found naturally downstream of Pfdx and upstream of the start codon. In our project we use the regulatory region for the fdx gene comprising Pfdx-t14c ([https://parts.igem.org/Part:BBa_K2992016 BBa_K2992016]) and this entry to regulate our volatile reporter genes for predicting the production of botulinum neurotoxin following food manufacture. <br><br>
  
 
===Characterisation===
 
===Characterisation===
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Data incoming
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One of the goals of goal of this experiment was to characterize the Pfdx promoter in a composite part  ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K2992043 BBa_K2992043]) with a new reporter protein, the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST, [https://parts.igem.org/wiki/index.php?title=Part:BBa_K2992000 BBa_K2992000]). The part was characterized through a fluorescence assay in <em>E.&nbsp;coli</em> as well as in <em>C.&nbsp;sporogenes</em>. Fluorescence is reported as Molecule Equivalent Fluorescence per Particle (MEFL/particle) as per the recommendation of the [https://2019.igem.org/Measurement/Protocols iGEM measurement Hub].
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It was assayed along with the following composite parts:
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- [https://parts.igem.org/wiki/index.php?title=Part:BBa_K2992044 BBa_K2992044]<br>
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- [https://parts.igem.org/wiki/index.php?title=Part:BBa_K2992042 BBa_K2992042]<br>
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- [https://parts.igem.org/wiki/index.php?title=Part:BBa_K2992012 BBa_K2992012] with this 5'UTR including a RBS [https://parts.igem.org/wiki/index.php?title=Part:BBa_K2992014  BBa_K2992014] and the FAST reporter protein [https://parts.igem.org/wiki/index.php?title=Part:BBa_K2992000 BBa_K2992000].<br>
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- [https://parts.igem.org/Part:BBa_K2715010 BBa_K2715010], the 5' UTR [https://parts.igem.org/Part:BBa_K2715019 BBa_K2715019],  this RBS [https://parts.igem.org/Part:BBa_K2715009 BBa_K2715009] and the FAST reporter protein [https://parts.igem.org/wiki/index.php?title=Part:BBa_K2992000 BBa_K2992000].<br>
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- [https://parts.igem.org/Part:BBa_J23106 BBa_J23106], the 5' UTR [https://parts.igem.org/Part:BBa_K2715019 BBa_K2715019],  this RBS [https://parts.igem.org/Part:BBa_K2715009 BBa_K2715009] and the FAST reporter protein [https://parts.igem.org/wiki/index.php?title=Part:BBa_K2992000 BBa_K2992000].<br>
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<br>
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https://2019.igem.org/wiki/images/5/5e/T--Nottingham--Basic4.png
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<br>
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https://2019.igem.org/wiki/images/6/6b/T--Nottingham--Basic3.png
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<br>
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The first observation from the expression of the FAST protein using different <em>Clostridium</em> and <em>E.&nbsp;coli</em> promoters is that FAST is a suitable reporter gene, both in <em>E.&nbsp;coli</em> and in <em>Clostridium&nbsp;sporogenes</em>. Indeed, quantifiable levels of fluorescence were recorded in between 6.3*10<sup>3</sup> MEFL/particle and 1.1*10<sup>6</sup> MEFL/particle. Pfdx is the strongest of the promoters tested in <em>E.&nbsp;coli</em> after Pthl, and the strongest in <em>C.&nbsp;sporogenes</em>.
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<br>
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For more characterisation details, please see the Results page.
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<br>
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https://2019.igem.org/Team:Nottingham/Results
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<br>
  
 
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===References===  
 
===References===  
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Cañadas et al., 2019 RiboCas - update
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RiboCas: A Universal CRISPR-Based Editing Tool for Clostridium. (2019). ACS Synthetic Biology, [online] p. Available at: https://pubs.acs.org/doi/abs/10.1021/acssynbio.9b00075 [Accessed 21 Oct. 2019].
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Minton et al., 2016 Road map - update
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Minton, N., Ehsaan, M., Humphreys, C., Little, G., Baker, J., Henstra, A., Liew, F., Kelly, M., Sheng, L., Schwarz, K. and Zhang, Y. (2016). A roadmap for gene system development in Clostridium. Anaerobe, 41, pp.104-112.
  
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<!-- Uncomment this to enable Functional Parameter display  
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2992017 parameters</partinfo>
 
<partinfo>BBa_K2992017 parameters</partinfo>
 
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Latest revision as of 20:08, 21 October 2019

5-UTR containing RBS for fdx gene from C. sporogenes

5’-UTR containing RBS for fdx gene from C. sporogenes


Usage and Biology

Ferredoxin is an iron-sulfur protein involved in central redox reactions occurring during the general metabolism of Clostridia. 5’-UTR and RBS region for fdx is found naturally downstream of Pfdx and upstream of the start codon. In our project we use the regulatory region for the fdx gene comprising Pfdx-t14c (BBa_K2992016) and this entry to regulate our volatile reporter genes for predicting the production of botulinum neurotoxin following food manufacture.

Characterisation

One of the goals of goal of this experiment was to characterize the Pfdx promoter in a composite part (BBa_K2992043) with a new reporter protein, the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST, BBa_K2992000). The part was characterized through a fluorescence assay in E. coli as well as in C. sporogenes. Fluorescence is reported as Molecule Equivalent Fluorescence per Particle (MEFL/particle) as per the recommendation of the iGEM measurement Hub.

It was assayed along with the following composite parts:

- BBa_K2992044
- BBa_K2992042
- BBa_K2992012 with this 5'UTR including a RBS BBa_K2992014 and the FAST reporter protein BBa_K2992000.
- BBa_K2715010, the 5' UTR BBa_K2715019, this RBS BBa_K2715009 and the FAST reporter protein BBa_K2992000.
- BBa_J23106, the 5' UTR BBa_K2715019, this RBS BBa_K2715009 and the FAST reporter protein BBa_K2992000.



T--Nottingham--Basic4.png
T--Nottingham--Basic3.png
The first observation from the expression of the FAST protein using different Clostridium and E. coli promoters is that FAST is a suitable reporter gene, both in E. coli and in Clostridium sporogenes. Indeed, quantifiable levels of fluorescence were recorded in between 6.3*103 MEFL/particle and 1.1*106 MEFL/particle. Pfdx is the strongest of the promoters tested in E. coli after Pthl, and the strongest in C. sporogenes.
For more characterisation details, please see the Results page.
https://2019.igem.org/Team:Nottingham/Results

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

RiboCas: A Universal CRISPR-Based Editing Tool for Clostridium. (2019). ACS Synthetic Biology, [online] p. Available at: https://pubs.acs.org/doi/abs/10.1021/acssynbio.9b00075 [Accessed 21 Oct. 2019].

Minton, N., Ehsaan, M., Humphreys, C., Little, G., Baker, J., Henstra, A., Liew, F., Kelly, M., Sheng, L., Schwarz, K. and Zhang, Y. (2016). A roadmap for gene system development in Clostridium. Anaerobe, 41, pp.104-112.