Team:Nottingham/Contribution


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Contribution


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Part
Contribution

Contribution

Characterisation of the constitutive promoter from C. sporogenes ferrodoxin gene [BBa_K2715011]


The part we sought to characterise was the constitutive promoter from C. sporogenes ferrodoxin gene (Pfdx), BBa_K2715011, used by the 2018 Nottigham iGEM team. We chose to measure expression of the Fluorescent reporter gene FAST (BBa_K2992000), comparing Pfdx against a range of reporters not yet used to characterise Pfdx. This included Pthl, PbotR, Pntnh, BBa_J23106 and a no-promoter control.


Figure 1. The various promoters used to compare the expression of the FAST protein for characterisation of Pfdx. Restriction sites varied with some promoters, though this did not affect the results of the characterisation.

Results

Figure 2. 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 Pfdx used experimentally throughout our project and Pfdx C14T (BBa_K2715011) are not significantly different, thus validating our experimental work.

Figure 3. 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 Pfdx used experimentally throughout our project and Pfdx C14T (BBa_K2715011) are not significantly different, thus validating our experimental work.

Figures 2 and 3 show the difference in promoter expression in E. coli and C. sporogenes respectively. 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 Pfdx used experimentally throughout our project and Pfdx C14T (BBa_K2715011) are not significantly different, thus validating our experimental work.


As described by Figures 2 and 3, it was found that Pfdx is a far more potent constitutive promoter in C. sporogenes than Pthl, PbotR, Pntnh, BBa_J23106 and the no-promoter control. However, expression in E. coli (Fig. 2) shows Pthl to be be a significantly stronger promoter. From this we can recommend the use of Pfdx as a strong constitutive promoter in Clostridial species.

BBa_K2715011 Contains a C14T Point Mutation

For quantitative characterisation, the expression of BBa_K2715011 and Bba_J23106 have been compared to a range of Clostridium promoters. Pfdx and Bba_K2715011 differ from only one base (C14T) 190 bp upstream of the Shine-Dalgarno sequence. As anticipated from such a benign mutation, the difference in expression levels between these two promoter is not significant (P=0.779 in C. sporogenes). We propose the C14T mutation in Bba_K2715011 should be reversed in order to avoid confusion regarding the sequence of the Pfdx promoter.


Pfdx and Bba_K2715011 have equivalent expression levels (5.5*105 +- 0.9*105 MEFL/particle and 6*105 +-1*105 MEFL/particle respectively in C. sporogenes [Fig. 1] ; 9*105 +- 1*105 105 MEFL/particle and 1.007*106 +-0.009*106 105 MEFL/particle respectively in E. coli [Fig. 2]). As such, Pfdx and Bba_K2715011 are interchangeable. However, since our part “Pfdx” is the original, unmutated version of the ferredoxin promoter from C. sporogenes, the sequence of Bba_K2715011 documented in the registry of parts should be curated to match the native sequence of Pfdx.

Use of BBa_J23106 for Low Expression of Genes in Clostridial Species

Additionally, BBa_J23106 is being expressed at significant levels (p<0.05) in C. sporogenes, with a normalised fluorescence of 6.3*104 +-1.3*104 MEFL/particle. Consequentially, this promoter could be used in the future for genes that require very low expression in C. sporogenes.