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| <section class="section"> | | <section class="section"> |
| <article> | | <article> |
− | <p style="text-align: justify; margin-bottom: 1em;"> | + | <div> |
− | Every Synbio Experiment is more or less based on the same principle: You change a system in some way and you | + | <figure style="float:right; margin-left: 25px;"> |
− | look at the outcome. This readout is one of the most important things in all natural science, a wrong readout
| + | <img style="height: 500px; width: 750px;" |
− | can easily flaw your whole experiment or can lead to serious misconclusion.
| + | src="https://static.igem.org/mediawiki/2019/4/4f/T--Marburg--Measurement-TeLUC%2BNanoluc.png" |
− | </p>
| + | alt="Comparison of Nanoluc, TeLuc luminescence spectra"> |
− | <p style="text-align: justify; margin-bottom: 1em;">
| + | <figcaption style="max-width: 550px; text-align: center"> |
− | The most common way to measure localisation, interaction or even the intensity of genetic elements is via
| + | Fig.1 - Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus |
− | Fluorescence as readout. Fluorescence Proteins (FP), started with the green fluorescent protein, are based on
| + | UTEX |
− | the ability of a chromophore to absorb photons of specific wavelength and emit this photon at another. Even on
| + | 2973 Absorption spectra. |
− | the iGEM registry, the characterization via FPs is the suggested way to characterise a part. This Method is
| + | </figcaption> |
− | prone to Background noise, depends on the folding of the Protein at the specific cell conditions and
| + | </figure> |
− | furthermore the chromophore can even bleach after to much exposure, so the drawbacks are obvious.
| + | |
− | </p>
| + | |
− | <p style="text-align: justify; margin-bottom: 1em;">
| + | |
− | 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.
| + | |
− | </p>
| + | |
− | <p style="text-align: justify; margin-bottom: 1em;">
| + | |
− | 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.
| + | |
− | </p>
| + | |
− | <figure style="float:right; margin-left: 25px;">
| + | |
− | <img style="height: 500px; width: 700px;"
| + | |
− | src="https://static.igem.org/mediawiki/2019/4/4f/T--Marburg--Measurement-TeLUC%2BNanoluc.png"
| + | |
− | alt="Comparison of Nanoluc, TeLuc luminescence spectra">
| + | |
− | <figcaption style="max-width: 550px; text-align: center">
| + | |
− | Fig.1 - Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus UTEX
| + | |
− | 2973 Absorption spectra.
| + | |
− | </figcaption>
| + | |
− | </figure>
| + | |
− | <p style="text-align: justify; margin-bottom: 1em;">
| + | |
− | 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.
| + | |
− | </p>
| + | |
− | <p style="text-align: justify; margin-bottom: 1em;">
| + | |
− | Driven by this problem, we dig ourselves in <a style="padding: 0"
| + | |
− | href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678970/">literature</a> and found a solution: A
| + | |
− | mutated
| + | |
− | Version of nanoLuc, so called <a style="padding: 0" href="http://parts.igem.org/Part:BBa_K3228042">teLuc</a>
| + | |
− | 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 | + | <p style="text-align: justify; margin-bottom: 1em;"> |
− | (D19S/D85N/C164H), which basically allows Diphenylterazine (DTZ) to prominently bind. This improved and | + | Every Synbio Experiment is more or less based on the same principle: You change a system in some way and you |
− | better part could catalyse a whole new and bright era of characterisation of Synthetic biology. | + | look at the outcome. This readout is one of the most important things in all natural science, a wrong |
− | </p>
| + | readout |
− | <p style="text-align: justify; margin-bottom: 1em;">
| + | can easily flaw your whole experiment or can lead to serious misconclusion. |
− | To demonstrate the redshift, transformed both <a style="padding: 0"
| + | </p> |
− | href="http://parts.igem.org/Part:BBa_K1159001">NanoLuc</a>and TeLuc under the Promoter 2_05 in e.coli. | + | <p style="text-align: justify; margin-bottom: 1em;"> |
− | This rather weak Promoter were chosen to showcase the ability of Luminescence to measure weak genetic
| + | The most common way to measure localisation, interaction or even the intensity of genetic elements is via |
− | elements. Both cells were grown to an OD600 of 0,8. After that a 1:100 dilution were used for the
| + | Fluorescence as readout. Fluorescence Proteins (FP), started with the green fluorescent protein, are based |
− | Measurements of the Luminescence spectra. The Results are summarized in Figure 2.
| + | on |
− | </p>
| + | the ability of a chromophore to absorb photons of specific wavelength and emit this photon at another. Even |
− | <figure style="float:right; margin-left: 25px;">
| + | on |
− | <img style="height: 500px; width: 700px;"
| + | the iGEM registry, the characterization via FPs is the suggested way to characterise a part. This Method is |
− | src="https://static.igem.org/mediawiki/2019/8/82/T--Marburg--TeLucandNanoLuc%2BUTEXSpectra.png"
| + | prone to Background noise, depends on the folding of the Protein at the specific cell conditions and |
− | alt="TeLuc and NanoLuc measurement in E.coli">
| + | furthermore the chromophore can even bleach after to much exposure, so the drawbacks are obvious. |
− | <figcaption style="max-width: 550px; text-align: center">
| + | </p> |
− | Fig.2 - Normalized Luminescence measurements of TeLuc and NanoLuc over their full spectra in e.coli.
| + | <p style="text-align: justify; margin-bottom: 1em;"> |
− | </figcaption>
| + | Bioluminescence could make the desired difference, but the original Luciferase Assays either consistent of |
− | </figure>
| + | an |
− | <p style="text-align: justify; margin-bottom: 1em;">
| + | whole Operon systems, or put an unnecessary high metabolic burden through ATP dependency and/or trough its |
− | We successfully showed the redshift of teluc in comparison to nanoLuc. This could will lead to a further | + | relatively large size (Firefly-Luciferase 61,5 kDa). Together with the low quantity, which can be several |
− | ~7 fold increase of Lumience in Cyanobacteria or plants.
| + | orders of magnitude lower than a fluorescence based system, the common breakthrough of Lumincese in |
− | Buy using our improve BioBrick for Luminescence Measurement, accurate and precise data can be obtained in
| + | Synthetic |
− | phototrophic Organism.
| + | biology is still missing. |
− | </p>
| + | </p> |
| + | <p style="text-align: justify; margin-bottom: 1em;"> |
| + | 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. |
| + | </p> |
| + | |
| + | <p style="text-align: justify; margin-bottom: 1em;"> |
| + | 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. |
| + | </p> |
| + | <figure style="float:right; margin-left: 25px;"> |
| + | <img style="height: 500px; width: 750px;" |
| + | src="https://static.igem.org/mediawiki/2019/8/82/T--Marburg--TeLucandNanoLuc%2BUTEXSpectra.png" |
| + | alt="TeLuc and NanoLuc measurement in E.coli"> |
| + | <figcaption style="max-width: 550px; text-align: center"> |
| + | Fig.2 - Normalized Luminescence measurements of TeLuc and NanoLuc over their full spectra in e.coli. |
| + | </figcaption> |
| + | </figure> |
| + | <p style="text-align: justify; margin-bottom: 1em;"> |
| + | Driven by this problem, we dig ourselves in <a style="padding: 0" |
| + | href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678970/">literature</a> and found a solution: A |
| + | mutated |
| + | Version of nanoLuc, so called <a style="padding: 0" href="http://parts.igem.org/Part:BBa_K3228042">teLuc</a> |
| + | 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. |
| + | </p> |
| + | <p style="text-align: justify; margin-bottom: 1em;"> |
| + | To demonstrate the redshift, transformed both <a style="padding: 0" |
| + | href="http://parts.igem.org/Part:BBa_K1159001">NanoLuc</a>and 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. |
| + | </p> |
| + | |
| + | <p style="text-align: justify; margin-bottom: 1em;"> |
| + | 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. |
| + | </p> |
| + | </div> |
| </article> | | </article> |
| </section> | | </section> |