Part:BBa_K2992000
FAST reporter gene
Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST) reporter gene.
Usage and Biology
FAST is one of the few fluorescent reporters available for effective use in anaerobic organisms. FAST is derived from Halorhodospira halophila and has been codon optimised for fluorescence studies in the genus Clostridium. In our project we use FAST as a reporter to demonstrate the activity of our chosen promoters and to assess its suitability as an alternative non-volatile reporter system for predicting neurotoxin production.
Characterisation
This part was used as part of our FAST reporter constructs - BBa_K2992042,BBa_K2992043,BBa_K2992044,BBa_K2992045, BBa_K2992046, BBa_K2992047,BBa_K2992048 - which were characterised using the FAST fluorometric assay.
Nottingham iGEM2019: Characterisation of FAST using different Clostridium and Escherichia coli promoters.
One of the goals of goal of this experiment was to characterize the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST). FAST is a protein that becomes fluorescent once bound to a fluorogenic ligand. Its main advantage is to be effective in absence of molecular oxygen (contrarily to standard fluorescent reporters such as GFP), which makes it compatible with work in Clostridium. It is also more convenient to use than gusA, the standard reporter used in Clostridium. The part was characterized through a fluorescence assay in E. coli as well as in C. sporogenes, along other promoters to assess their relative strength. Fluorescence is reported as Molecule Equivalent Fluorescence per Particle (MEFL/particle) as per the recommendation of the iGEM measurement Hub.
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. However, it is worth noting, the assembly of the strong promoter BBa_J23119 with the FAST gene only produced colonies with heavily mutated BBa_J23119, indicating that FAST expression could be toxic under the control of such a strong promoter. It is however somewhat surprising that Pthl was not also mutated, as last year’s [http://2018.igem.org/Team:Nottingham/Collaborations#warwick-and-imperial interlab study].
For more characterisation details, please see the Results page.
https://2019.igem.org/Team:Nottingham/Results
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 155
References
Streett, H., Kalis, K. and Papoutsakis, E. (2019). A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST). Applied and Environmental Microbiology, 85(14).
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