Difference between revisions of "Part:BBa K2992000"

Latest revision as of 22:49, 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

Characterisation of FAST using different Clostridium and Escherichia coli promoters.

The part was characterized in a fluorescence assay in E. coli as well as in C. sporogenes, through associations with 6 different promoters, and two different 5'UTR and RBS. It was included in the following composite parts:

    - Pfdx-FAST (BBa_K2992043) 
    - PntnH-FAST: BBa_K2992044
    - No promoter-FAST: BBa_K2992042

FAST was also flanked by the following basic promoter parts upstream of the Clostridium acetobutylicum thl 5'UTR (BBa_K2715019) and RBS (BBa_K2715009):

    - Pfdx_C14T: Bba_K2715011
    - Pthl: BBa_K2715010
    - BBa_J23106

And followed by the Tfdx terminator (BBa_K2284012).

At last, it was flanked by this basic promoter part associated with the Clostridium botulinum botR 5'UTR and RBS (BBa_K2992014):

    - PbotR: BBa_K2992012

And followed by the Tfdx terminator (BBa_K2284012).


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. .

Use of FAST in a BotR reporter system in C. sporogenes BotR Strains

This part was used as part of our FAST reporter constructs - Promoterless FAST reporter constructBBa_K2992042, FAST reporter construct with Pfdx 5-UTR+RBSBBa_K2992043, FAST reporter construct with PntnH 5-UTR+RBS BBa_K2992044, FAST reporter construct with Pthl 5-UTR+RBS BBa_K2992045, FAST reporter construct with PbotR 5-UTR+RBS BBa_K2992046, FAST reporter construct with Pha33 5-UTR+RBS BBa_K2992047, FAST reporter construct with Pj23106 and thl RBS BBa_K2992048 - which were characterised using the FAST fluorometric assay.

Considerable FAST activity was detected when botR was expressed from the genome of C. sporogenes using its native promoter when coupled with plasmid-borne PntnH-FAST (red lines). Crucially, the level of FAST detected in the absence of genomic botR was comparable to the promoter-less FAST plasmid (green vs orange lines). Plasmid-borne Pfdx-FAST generated sufficient reporter activity across all time-points. These data demonstrate that genomic botR can be used to regulate reporter gene expression to appreciable levels when placed under the control of the PntnH promoter. The FAST data corroborates the data obtained from our acetone production experiments, thus consolidating the suitability of our C. sporogenes reporter strains as models for the prediction of Botulinum neurotoxin production.

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).