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Improve

NanoLuc-mNeonGreen (BBa_K3168009)

Comparing mNeonGreen (BBa_K1761003) & NanoLuc-mNeonGreen (BBa_K3168009)

Fluorescent labeling has become an effective tool for intracellular imaging. Even though fluorescent sensors are currently important tools, there are several limitations associated with them. Fluorescent sensors are external illumination-dependent, which limits measurements over extended periods of time due to photobleaching and phototoxicity. Furthermore, autofluorescence and light scattering impedes measurements in media such as blood [1].

Bioluminescence has overcome these limitations because light is produced through a chemical reaction instead of external illumination. Bioluminescence is generated when a photon-emitting substrate, called luciferin, is oxidized. This process is catalyzed by an enzyme called luciferase. NanoLuc (BBa_K1159001) is one example of a luciferase and emits blue light upon addition of the substrate furimazine (Figure 1).

Figure 1: Illustration of the oxidation of furimazine catalyzed by the luciferase NanoLuc [2].

mNeonGreen (BBa_K1761003) is a yellow-green fluorescent protein that can be excited with a wavelength of 480 nm. The excitation spectrum of mNeonGreen overlaps with the emission spectrum of NanoLuc, which has a maximum emission of 460 nm. By combining the donor NanoLuc and the acceptor mNeonGreen (BBa_K3168009), bioluminescence resonance energy transfer (BRET) occurs when these two are in close proximity (Figure 2). This means that no further external illumination is needed to excite mNeonGreen, which decreases autofluorescence and light scattering. Therefore, the addition of NanoLuc to create NanoLuc-mNeonGreen improves mNeonGreen because no laser external illumination is needed while maintaining the functionality of mNeonGreen.

Figure 2: Schematic representation of BRET between the donor NanoLuc and the acceptor mNeonGreen.

This concept was shown to function as expected for varying concentrations of NanoLuc-mNeonGreen, when its light intensity was measured upon addition of furimazine and without external illumination (Figure 3).

Figure 3: BRET spectrum NanoLuc-mNeonGreen.

Comparing NanoLuc (BBa_K1159001) & NanoLuc-mNeonGreen (BBa_K3168009)

Furthermore, NanoLuc-mNeonGreen is also an improved part compared to only NanoLuc. First of all, NanoLuc-mNeonGreen allows for bioluminescent measurements at other wavelengths than NanoLuc and thus for the detection of more than one component at the same time. In addition, it generates higher intensities than NanoLuc alone, about four and a half times as high (Figure 4).

Figure 4: Luminescence intensity at emission maximum for NanoLuc (460 nm) and NanoLuc-mNeonGreen (517 nm) for increasing concentrations of both proteins.

Furthermore, NanoLuc alone gives one signal, which is disadvantageous because a single signal can be influenced by many factors. Therefore, it is better to perform ratiometric measurements. NanoLuc-mNeonGreen grants ratiometric measurements where NanoLuc-mNeonGreen acts as a calibrator luciferase, which allows time and concentration independent measurements. NanoLuc-mNeonGreen thus offers a whole new range of measurements as opposed to normal NanoLuc. Usage of NanoLuc-mNeonGreen as calibrator luciferase is described in more detail on our Measurement page.

Improvement NanoLuc (BBa_K1159001)

We developed a cysteine-free version of NanoLuc (BBa_K3168006) which is an improvement of the original version of NanoLuc (BBa_K1159001). By mutating the cysteine to a serine, the luciferase can be used in fusion proteins that need to be labeled through maleimide coupling, such as our own dCas9-BRET sensor protein (BBa_K3168007).

Comparing Cysteine-free-NanoLuc & NanoLuc

After successful expression and purification of both NanoLuc proteins, measurements could be performed to determine whether cysteine-free-NanoLuc was still functional and could be used as a replacement for NanoLuc in fusion proteins where subsequent maleimide coupling is required. Bioluminescence intensity was measured for different concentrations of both NanoLuc proteins (Figure 5). This shows that cysteine-free-NanoLuc has a similar shape of the emission spectrum and has its maximum at the same wavelength as NanoLuc. It furthermore shows that cysteine-free-NanoLuc is still functional, although showing a decrease of about fifty percent in intensity for all measured concentrations. However, due to NanoLuc’s initial brightness, cysteine-free-NanoLuc is still a lot brighter than most other luciferases while also maintaining its small size and high stability [2, 3].

Figure 5: Emission spectrum (A) and maximum intensity at 460 nm (B) of Cysteine-free-NanoLuc and NanoLuc.

To conclude, we have developed an improvement of NanoLuc that is still highly functional and can be used in a wider field of applications, such as in fusion proteins when one wishes to label the fusion protein through maleimide coupling.

Improvement paired dCas9 reporter Peking 2015 (BBa_K1689009 & BBa_K1689010)

Peking 2015 has developed a paired dCas9 reporter by combining dCas9 and split Firefly luciferase to detect specific DNA targets. Their reporter consists of two parts, a cluc-dCas9 (BBa_K1689009) and an nluc-dCas9 (BBa_K1689010), that together form the paired dCas9 reporter. However, the Firefly luciferase generates a low light intensity and is therefore not considered a lot for bioluminescent labeling. This has changed with the recent development of the deep-sea shrimp derived luciferase NanoLuc [3]. The new luciferase NanoLuc is smaller compared to the Firefly luciferase and therefore offers certain advantages over the traditional methods. NanoLuc has increased stability, smaller size and a >150-fold increase in bioluminescence [2]. Furthermore, NanoLuc displays high physical stability, maintains its activity during incubation up to 55 °C or in culture medium for >15 h at 37 °C and shows no evidence of posttranslational modifications or subcellular partitioning in mammalian cells [3]. Furimazine, NanoLuc’s substrate, shows increased stability and lower background activity, which enhances the possibilities for bioluminescence imaging [2].

We, iGEM TU_Eindhoven, have therefore developed a new paired dCas9 reporter with Split-NanoLuc (BBa_K3168004 & BBa_K3168005) to obtain a lower limit of detection to allow for a wider range of applications. Due to the unavailability of the DNA of the Peking sensor, we were unable to express the sensor and compare our own measurement data from their sensor with data from our improved sensor. Therefore, we compared the results from our improved sensor with the data published by Peking (Figure 6).

Figure 6: A) Limit of detection for dCas9 split firefly luciferase as reported by Peking 2015 (BBa_K1689009 & BBa_K1689010). B) Limit of detection for our paired dCas9-Split-NanoLuc system.

Whereas the limit of detection of Peking 2015 with the split firefly was determined to lie at 100 pM, we have been able to do reliable detection of target DNA at concentrations of 10 pM with our paired dCas9-Split-NanoLuc system. This means that we have improved the dCas9 split luciferase system by incorporating split-NanoLuc instead of the split Firefly and have thereby improved the detection limit by at least a tenfold. It is even expected that the limit of detection for our system lies lower than the 10 pM presented because this limit was determined in sub-optimal conditions. I.e. the measurements were performed with an interspace distance of 20 base pairs while other measurements have shown that the optimal interspace distance lies at 70 basepairs (see Results).

Furthermore, this limit of detection was determined with dCas9-Split-NanoLuc recognizing one site on one target. Multiple recognition sites on one target for dCas9-Split-NanoLuc even resulted in a limit of detection of 5 pM, thus a twentyfold improvement in comparison with the sensor by Peking (Figure 7). Other measurements performed to validate the functioning of our sensor are described at Results.

Figure 7: Limit of detection for paired dCas9-Split-NanoLuc (2 nM) system with multiple recognition sites on one target.

References

Arts, R., Aper, S. J., & Merkx, M. (2017). Engineering BRET-Sensor Proteins. In Methods in enzymology (Vol. 589, pp. 87-114). Academic Press.
England, C. G., Ehlerding, E. B., & Cai, W. (2016). NanoLuc: a small luciferase is brightening up the field of bioluminescence. Bioconjugate chemistry, 27(5), 1175-1187.
Hall, M. P., Unch, J., Binkowski, B. F., Valley, M. P., Butler, B. L., Wood, M. G., ... & Robers, M. B. (2012). Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS chemical biology, 7(11), 1848-1857.

Team:TU Eindhoven/Improve