Latest revision as of 16:09, 18 September 2015

Harmonised B. subtilis Falvin Binding Fluorescent Protein (BsFP)

Gblock design of BsFP gene includes the ORF, B.subtilis RBS and an N-terminal HisTag

Background

The Bacillus subtilis flavin-binding fluorescent protein (BsFbFP, or BsFP) is native to the ‘’B. subtilis’’ bacteria, and homologs are also found in several other bacterial genera. This flavin-binding fluoroprotein has a different chromophore to the more widely used GFP family of fluoroproteins, and BsFP has several advantages over the more-traditional fluoroproteins such as GFP (BBa_E0040). The BsFP gene is much smaller than GFP (around 700 bp vs 137 bp), and it is capable of folding and fluorescing under anaerobic conditions, unlike GFP and its homologs. The BsFP folds into a fluorescent form faster and more efficiently ¹. Our development of this part represents an improvement of function and characterisation of similar parts already existing in the iGEM registry, such as BBa_K376004, BBa_K1094000 and BBa_K660000.

Experimental Validation

In our project, we used the BsFP protein to experimentally validate our novel codon harmonisation algorithm, [http://2015.igem.org/Team:Sydney_Australia/TransOpt TransOpt]. We were inspired to choose this protein as our model system based on the work of previous iGEM teams, who developed related parts BBa_K376004, BBa_K1094000 and BBa_K660000. We tried to improve the function of BsFP by utilising three different codon-optimisation / harmonisation approaches; these were our novel 'in-house' algorithm TransOpt, the standard harmonisation methods proposed by Angov et al. ², and a ‘fast-folding’ method (more information on our team's [http://2015.igem.org/Team:Sydney_Australia/TransOpt TransOpt page]).

Our model was hypothesised based on previous literature demonstrating that the ribosome translation kinetics profile was influenced by both the copy number of each specific tRNA gene in the host organism, and by the codon redundancy, and that these two factors were paramount in controlling protein folding, which in turn yields protein activity ³. In this experimental validation, we performed fluorescence assays to detect the correctly-folded proteins, with the assumption being that higher fluorescence is characteristic of a better folded protein. We generated the following four sequences:

• BsFP-WT: native sequence from the native host B. subtilis
• BsFP-fast: all codons were replaced with the E.coli versions that possessed the fastest translation rate, as quantised using the approach of Spencer et al ³
• BsFP-standard: harmonised BsFP generated via standard harmonisation (as proposed by Angov et al ²) using the rate quantisation approach of Spencer et al ³
• BsFP-TransOpt: optimised BsFP sequence generated using our new TransOpt algorithm

Each variant of BsFP was cloned into a tetracycline-inducible expression vector (pUS212), transformed into E.coli, and clones confirmed to carry the correct constructs were induced with tetracycline, and then both fluorescence (520 nm) and optical desnity (600 nm) were measured in triplicate samples of each culture. The fluorescence data were normalised by dividing by the optical density and subtracting the fluorescence from the E. coli pUS212, containing no BsFP gene.

After performing the fluorescence assay, we found that the BsFP subjected to standard harmonisation possessed the highest fluorescence, which was 2 fold higher than the wild-type sequence. The fast-folding and Trans-Opt versions of the gene were less fluorescent than wild type. These data was also qualitatively confirmed by exposing tet-induced patches on plates to a long-wave UV lamp; only the standard-harmonisation clones yielded fluorescence visible with the naked eye.

Graphs showing the fluorescence and relative fold change in fluorescence (compared to BsFP WT) at 520 nm and 500 nm emission after 460 nm excitation.

Plate containing tet-induced E. coli JM109 cells expressing different versions of the BsFP protein, or a vector-only control (no BsFP). Note that WT stands for wild type.

Although we failed to demonstrate that the TransOpt algorithm facilitated greater fluorescence in the case of BsFP, we predict that this approach may still be useful for other proteins. Due to its poor fluorescence, we did not submit the TransOpt-harmonised BsFP as a Part. Instead, we submit the standard-harmonisation BsFP gene to the Registry.

Part Description

The part submitted herein is BsFP optimised using a standard harmonisation methodology (as proposed by Angov et al ²) using the rate quantisation approach of Spencer et al. ³ As our experimental validation has shown, this part functions as intended, and provides fluorescence levels more than a factor of two greater than the WT form. This Part is a versatile and widely-applicable alternative to GFP/RFP type fluoroproteins, due to its high fluorescence, small size, fast maturation, ad oxygen-independence.

This new BsFP Part has the potential to enhance reporter gene assays, improve biomarker tracking in cell tissues, create fluorescent fusion proteins, and many other applications. It is also appropriate to note that the gene encoding the protein is substantially different to the previously submitted parts BBa_K376004, BBa_K1094000 and BBa_K660000; while these cannot be directly compared in terms of fluorescence intensity, we note that our part is the most rigorously characterised of these. In particular, the ability to easily detect its fluorescence on agar plates is especially convenient.

If you would like to utilise our codon optimisation tools which were used to generate all BsFP variants for this part's experimental validation, refer to our modelling [http://2015.igem.org/Team:Sydney_Australia/Modeling/Supplementary supplementary materials] page for instructions on downloading and using the algorithm.

Structure of pSB1C3-BsFP plasmid construct submitted to the registry.

Sequence and Features

References

¹ Mukherjee, A., et al., Characterization of flavin-based fluorescent proteins: an emerging class of fluorescent reporters. PLoS One, 2013. 8(5): p. e64753.

² Angov E et al., 2008, “Heterologous Protein Expression Is Enhanced by Harmonizing the Codon Usage Frequencies of the Target Gene with those of the Expression Host”, PLoS One, Vol 3.

³ Spencer P et al., 2012, “Silent Substitutions Predictably Alter Translation Elongation Rates and Protein Folding Efficiencies”, Vol 422, pp. 328-335.