Part:BBa_K2992017

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.

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.