Difference between revisions of "Part:BBa K2992000"

(Characterisation)
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===Characterisation===
 
===Characterisation===
This part was used as part of our FAST reporter constructs - [https://parts.igem.org/Part:BBa_K2992042 BBa_K2992042],[https://parts.igem.org/Part:BBa_K2992043 BBa_K2992043],[https://parts.igem.org/Part:BBa_K2992044 BBa_K2992044],[https://parts.igem.org/Part:BBa_K2992045 BBa_K2992045],
 
[https://parts.igem.org/Part:BBa_K2992046 BBa_K2992046], [https://parts.igem.org/Part:BBa_K2992047 BBa_K2992047],[https://parts.igem.org/Part:BBa_K2992048 BBa_K2992048] - which were characterised using the FAST fluorometric assay.
 
  
 
===Characterisation of FAST using different <em>Clostridium</em> and <em>Escherichia coli</em> promoters.===
 
===Characterisation of FAST using different <em>Clostridium</em> and <em>Escherichia coli</em> promoters.===

Revision as of 22:16, 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 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.


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. 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. .


Characterisation of FAST in C. sporogenes BotR Strains

FAST curve.PNG


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


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
    INCOMPATIBLE 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).