Every Synbio Experiment is more or less based on the same principle: You change a system in some way and you look at the outcome. This readout is one of the most important things in all natural science, a wrong readout can easily flaw your whole experiment or can lead to serious misconclusion.

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 to much exposure, so the drawbacks are obvious.

Bioluminescence could make the desired difference, but the original Luciferase Assays either consistent of an whole Operon systems, or put an unnecessary high metabolic burden through ATP dependency and/or trough 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 Lumincese in Synthetic biology is still missing.

Newly developed small ATP independent Lucferase Proteins, are interesting candidates to bypass these Problems. Nanoluc, with its 19 kDa and up to 150 fold increase in brightness compared to the Firefly-Luciferase is handled as an suitable alternative. This Protein use the patented Substrate Furimazine, and emits Photons at 460 nm. Nanoluc has been successfully implemented in Promoter testing and as an alternative in Interaction messurement via Bilumiecnce Resonace energy transfer, but sadly only few team ever used this system.

One scratch on the surface of Nanoluc is for sure the restriction of the wavelength. While for Measurements in many organisms and Tissues, this looming Problem did not occur, it's becoming obvious, when looking into phototrophic Organisms and deep-tissue mammalian cells. 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. Chlorophyll a have one their two peaks at 440 nm [fig.1]. If one would compare that with the nanoluc spectra, a devastating conclusion could be made: The Photosystem will absorb photons from the Signal, leading to weaker peaks, and maybe more grave/frightening/alarming, a dependency of Signal on the chlorophyll content. Althroug localisation experiments should´t be affected that much, Measurement and characterisation, the foundation of which synthetic Biology is build on, could be shaken.

Driven by this problem, we dig ourselves in literature and found a solution: A mutated Version of nanoLuc, so called teLuc which has a severe red shifted pattern with a peak at 502 nm (Figure 2). What is even more serviere is the astonishing brightness, wich even surpass nanoluc by several folds (5,7) in vitro. In vivo this effect is even more dramatic, through its ability to bypass the absorption of Light. 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 3 Amino Acid changes in 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, transformed both NanoLucand TeLuc under the Promoter 2_05 in e.coli. This rather weak Promoter were chosen to showcase the ability of Luminescence to measure weak genetic elements. Both cells were grown to an OD600 of 0,8. After that a 1:100 dilution 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 could will lead to a further ~7 fold increase of Lumience in Cyanobacteria or plants. Buy using our improve BioBrick for Luminescence Measurement, accurate and precise data can be obtained in phototrophic Organism.