Team:Nottingham/Basic Part

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Best

Basic Part

Introducing the FAST Reporter Gene

Best

Basic Part

Best New Basic Part

FAST Reporter Gene

BBa_K2992000

For the Best New Basic Part Award, we would like to nominate Part BBa_K2992000: the FAST reporter gene. This new part is the coding sequence for the newly engineered fluorogen “Fluorescence-Activating and Absorption-Shifting Tag Protein” (FAST)^[1] produced by The Twinkle Factory.

We believe FAST to be a powerful new tool in the iGEM registry, as it is one of a small minority of fluorescent labels that can be used in low-oxygen and anaerobic environments. In our experiments, FAST yielded a very large expression range (a maximum 100-fold difference was observed in the present experiments); this renders it a sufficiently sensitive reporter suitable for promoter libraries, or other studies that may be interested in the elucidation of small expression differences between different constructs.

We have made use of this fluorogen as an alternative reporter to our volatile acetone reporter, and for the characterisation of the constitutive promoter from C.sporogenes ferrodoxin gene (P_fdx),BBa_K2715011 as the protocol for fluorescent assay is much faster than for other reporters, eg. GusA.

Results

Use of FAST as an Alternative Reporter in C. sporogenes

Figure 1. Optical Density at 600 nm (OD₆₀₀) of all the engineered C. sporogenes harbouring the FAST reporter constructs, over 48 hours. OD₆₀₀ was measured for all constructs grown in TYG supplemented with 30 mM glucose, measurements were taken at 0, 4, 8, 24 and 48 hours.

Figure 2. Normalised fluorescence of the engineered C. sporogenes strains harbouring theFASTreporter constructs, over 48 hours. Three biological replicates of the strain were grown in TYG supplemented with 30 mM glucose, with 1 ml samples taken at 8, 24 and 48 hour timepoints. Relative fluorescence units (RFU) was normalised by dividing the RFU by the respective OD₆₀₀ values at each timepoint.

We found that inserting a plasmid containing the FAST coding gene in C. sporogenes, in constructs with and without the native sigma factor P_botR, resulted in a growth curve (measured by optical density) similar to that of wild type sporogenes. This indicates that FAST is non-toxic and does not inhibit growth in C. sporogenes. The construct containing the sigma factor P_botR produced the highest relative fluorescence, validating our use of P_botR in our final reporter strain.

Use of FAST to Characterise P_fdx in C. sporogenes and E. coli

Figure 3. The difference in promoter expression in E. coli. This was measured by fluoresence normalised by optical density (OD), and converted to Fluorescence expressed in Molecule equivalent of Fluorescein per Particle (Cospheric Monodisperse Silica Microspheres 0.961um diameter. It should be noted that expression in the P_fdx used experimentally throughout our project and P_fdx C14T (BBa_K2715011) are not significantly different, thus validating our experimental work.

Figure 4. The difference in promoter expression in C. sporogenes. This was measured by fluoresence normalised by optical density (OD), and converted to Fluorescence expressed in Molecule equivalent of Fluorescein per Particle (Cospheric Monodisperse Silica Microspheres 0.961um diameter. It should be noted that expression in the P_fdx used experimentally throughout our project and P_fdx C14T (BBa_K2715011) are not significantly different, thus validating our experimental work.

FAST appears to be a suitable reporter gene, in both in E. coli and in Clostridium sporogenes as quantifiable levels of fluorescence were recorded between 6.3*10⁴ MEFL/particle and 1.1*10⁶ MEFL/particle. However using the strong constitutive promoter BBa_J23119 with FAST appeared to be toxic in E. coli. More information about this experiment can be found on our Contribution page.

Toxicity of FAST in E. coli

It should be noted that 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 interlab study) consistently found that Pthl was a stronger E coli promoter than PJ23119. It was for this reason that BBa_J23119 has been removed from our results as no usable data was collected.

References

1. 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).

With special thanks to: The Twinkle Factory

iGEM Nottingham 2019