With our limited understanding of the natural world, we are often dependent on experimentally deriving knowledge of complex system by analyzing how they change given certain alternations. Based on this principle, it is of utmost importance that the collected data are as accurate as possible, since a wrong readout can easily lead to a drastically different conclusion to an experiment. This year's team has gone to great lengths to carefully examine the currently used readout methods (also see fluorescence reporter and characterization of parts) and worked on improving them to counteract potential issues in order to further refine the field of Synthetic Biology.

The most common way to measure localisation, interaction or even the intensity of genetic elements is via fluorescence as readout. Fluorescence proteins (FP), started with the green fluorescent protein, are based on the ability of a chromophore to absorb photons of specific wavelength and emit this photon at another. Even on the iGEM registry, the characterization via FPs is the suggested way to characterise a part. This method is prone to background noise, depends on the folding of the protein at the specific cell conditions and furthermore the chromophore can even bleach after too much exposure, so the drawbacks are obvious.

Comparison of Nanoluc, TeLuc luminescence spectra
Fig.1 - Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus UTEX 2973 Absorption spectra. Note: This illustration doesn´t show real proportions.

Bioluminescence could make the desired difference, as luminescence doesn't require excitation, which lead to higher background noises. Especially in phototrophic organisms, where light is absorbed at a regular basis, this is a huge benefit. But original luciferase assays either consisting of a whole a operon system, or implementing an unnecessary high metabolic burden through ATP dependency and/or through its relatively large size (Firefly-Luciferase 61,5 kDa). Together with the low quantity, which can be several orders of magnitude lower than a fluorescence based system, the common breakthrough of luminescence in Synthetic Biology is still missing.

Newly developed small ATP independent luciferase proteins, are interesting candidates to bypass these problems (England et al., 2016). Nanoluc, with its 19 kDa and up to 150 fold increase in brightness compared to the Firefly-Luciferase proves to be a suitable alternative. This protein uses the patented substrate furimazine, and emits photons with a peak at 460 nm. Nanoluc has been successfully implemented in promoter testing (Oh-hashi et al., 2016), and as an alternative in interaction measurement via Bioluminescence Resonance Energy Transfer (BRET), but sadly only few teams ever used this system.

A huge drawback of NanoLuc (BBa_K1159001) is the restriction of the wavelength spectrum, which is rather low with 460 nm. This problem didn't occur in most organisms or tissues, however when working with phototrophic organisms or measuring deep-tissue mammalian cells there is a noticable drop in accuracy of protein expression to luminescence output (Yeh et al., 2017). As the keen reader might guess, cells absorb light of the wavelength under 600 nm to a great extent and even more if they have a photosystem. Cyanobacteria absorb light during photosynthesis, with one of their two peaks at 440 nm (Chlorophyll A) [fig.1]. As NanoLuc shows it maximal absorption at exactly that position, it is not best suited for measuring with protein expression output in cyanobacteria. Although localisation experiments should´t be affected that much, measurement and characterisation, the foundation of which Synthetic Biology is build on, are not very accurate.

TeLuc and NanoLuc measurement in E.coli
Fig.2 - Normalized Luminescence measurements of TeLuc and NanoLuc over their full spectra in E. coli.

Driven by this problem, we dig ourselves in literature (Yeh et al., 2017) and found a solution: A mutated Version of NanoLuc, so called teLuc (BBa_K3228042) which has a severe red shifted pattern with a peak at 502 nm (Figure 2). Even better is the reported astonishing brightness, which even surpassed NanoLuc by several folds (5,7x) in vitro. In vivo this effect is even more dramatic, through its ability to bypass the absorption of light by the cell (noticeable luminescence at >600 nm). We expect this ability of teLuc to surpass the limits of luminescence in plants to an amazing extent, and allow the plant Synthetic Biology community to accelerate their research. teLuc differs from its deep-sea origin ortholog only in three amino acid changes in the substrate binding pocket (D19S/D85N/C164H), which basically allows diphenylterazine (DTZ) to prominently bind. This improved and better part could catalyse a whole new and bright era of characterisation of Synthetic Biology.

To demonstrate the redshift, we transformed both NanoLuc (BBa_K1159001) and teLuc (BBa_K3228042) under the Anderson-Promoter BBa_J23103, in E.coli. This rather weak promoter was chosen to showcase the ability of luminescence to measure weak genetic elements, which is a problem for fluorescence reporters, due to high background noise. Both cells were grown to an OD600 of 0,8 and non-induced samples were used for normalisation. After a 1:100 dilution, 10 µl were used for the measurements of the luminescence spectra. The results are summarized in Figure 2.

We successfully showed the redshift of teLuc in comparison to NanoLuc. This alone will lead to a further ~7 fold increase of luminescence in cyanobacteria or plants. By using our improved BioBrick for luminescence measurement, accurate and precise data can be obtained in phototrophic organism.