Difference between revisions of "Part:BBa K2992000"

Revision as of 21:14, 21 October 2019

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.

Characterisation of FAST using different Clostridium and Escherichia coli promoters.

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] consistently found that Pthl was a stronger E coli promoter than PJ23119. .

For more characterisation details, please see the Results page.
https://2019.igem.org/Team:Nottingham/Results

Sequence and Features

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).