Difference between revisions of "Team:Marburg/Improve"

 
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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.
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The most common way to measure localisation, interaction or even the intensity of genetic elements is via Fluorescence as readout.
+
<div>
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.
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  <div class="box-dark">
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.
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    <!--titel der seite-->
 +
    <h1 class="heading">
 +
      I M P R O V E
 +
    </h1>
 +
    <hr class="line">
 +
    <img src="https://static.igem.org/mediawiki/2019/a/ac/T--Marburg--logo.svg" class="logo" alt="Syntex Logo">
 +
  </div>
 +
  <div style="margin-top: 11vh;">
 +
    <section class="section">
 +
      <article>
 +
        <div>
  
  
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 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. Naoluc 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.  
+
          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 <a style="padding: 0" href=" https://2019.igem.org/Team:Marburg/Design#toolbox" target="_blank"> fluorescence reporter and characterization of parts</a>) and worked on improving them to counteract potential issues in order to further refine the field of Synthetic Biology.
 +
          </p>
 +
          <p style="text-align: justify; margin-bottom: 1em;">
 +
            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 <a style="padding: 0"
 +
              href="https://parts.igem.org/Measurement/RPU/Measure" target="_blank">part</a>. 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.
 +
          </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="Comparison of Nanoluc, TeLuc luminescence spectra">
 +
            <figcaption style="max-width: 750px"; text-align: justify>
 +
              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.
 +
            </figcaption>
 +
          </figure>
 +
          <p style="text-align: justify; margin-bottom: 1em;">
 +
          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 <a style="padding: 0" href=" https://parts.igem.org/Lux#Lux_operon " target="_blank">operon system</a>, 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.
 +
          </p>
 +
          <p style="text-align: justify; margin-bottom: 1em;">
 +
            Newly developed small ATP independent luciferase proteins, are interesting candidates to bypass these
 +
            problems <a style="padding: 0" href=" https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4871753/" target="_blank"> (England <i>et al.</i>, 2016)</a>.
 +
            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 <a style="padding: 0" href=" https://onlinelibrary.wiley.com/doi/full/10.1111/gtc.12401" target="_blank"> (Oh-hashi <i>et al.</i>, 2016)</a>, and as an alternative in interaction
 +
            measurement via Bioluminescence Resonance Energy Transfer <a style="padding: 0" href=" https://link.springer.com/protocol/10.1007%2F978-1-4939-3673-1_17" target="_blank"> (BRET)</a>, but sadly only few teams ever used this system.
 +
          </p>
  
 +
          <p style="text-align: justify; margin-bottom: 1em;">
 +
            A huge drawback of <a style="padding: 0"
 +
              href="http://parts.igem.org/Part:BBa_K1159001" target="_blank">NanoLuc (BBa_K1159001)</a> 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 <a style="padding: 0"
 +
              href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678970/" target="_blank">(Yeh <i>et al.</i>, 2017)</a>. 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.
 +
          </p>
 +
          <figure style="float:right; margin-left: 25px;">
 +
            <img style="height: 500px; width: 750px;"
 +
              src="https://static.igem.org/mediawiki/2019/4/4f/T--Marburg--Measurement-TeLUC%2BNanoluc.png"
 +
              alt="TeLuc and NanoLuc measurement in E.coli">
 +
            <figcaption style="max-width: 750px"; text-align: justify>
 +
              Fig.2 - Normalized Luminescence measurements of TeLuc and NanoLuc over their full spectra in <i>E. coli</i>.
 +
            </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/" target="_blank">literature (Yeh <i>et al.</i>, 2017)</a> and found a solution: A
 +
            mutated
 +
            Version of NanoLuc, so called <a style="padding: 0" href="http://parts.igem.org/Part:BBa_K3228042" target="_blank">teLuc (BBa_K3228042)</a>
 +
            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.
  
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.
+
            teLuc differs from its deep-sea origin ortholog only in three amino acid changes in the substrate binding pocket
Driven by this problem, we dig ourselves in literature and found a our solution. We found a mutated Version of NanoLuc, so called teLuc, which has a severe red shifted pattern with a peak at 502 nm (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678970/)(figure). 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 thier reaseach.
+
            (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>
((((Bildunterschrieft: Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus UTEX 2973 Absorption spectra.)))))
+
          <p style="text-align: justify; margin-bottom: 1em;">
 
+
            To demonstrate the redshift, we transformed both <a style="padding: 0"
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.  
+
              href="http://parts.igem.org/Part:BBa_K1159001" target="_blank">NanoLuc (BBa_K1159001)</a> and <a style="padding: 0" href="http://parts.igem.org/Part:BBa_K3228042" target="_blank">teLuc (BBa_K3228042)</a> under the  Anderson-Promoter <a style="padding: 0" href="http://parts.igem.org/Part:BBa_J23103" target="_blank">BBa_J23103</a>, in <i>E.coli</i>.
This improved and better part could catalyse a whole new and bright era of characterisation of Synthetic biology.  
+
            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
To demonstrate the redshift, transformed both NanoLuc (http://parts.igem.org/Part:BBa_K1159001) 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.
+
            measurements of the luminescence spectra. The results are summarized in Figure 2.
Messume
+
          </p>
 
+
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.  ___
+
 
+
 
+
 
+
 
+
 
+
 
+
 
+
 
+
 
+
 
+
 
+
 
+
 
+
 
+
 
+
 
+
 
+
 
+
 
+
 
+
 
+
 
+
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. (Example, finde keins)  
+
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 this 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 expouserr, 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 (((How bright is this fucccccker )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. Naoluc 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 our solution. We found a mutated Version of NanoLuc, so called teLuc, which has a severe red shifted pattern with a peak at 502 nm (figure). What is even more serviere is the astonishing brightness, wich even surpass nanoluc by several folds (7.5) 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, dou to the lack of phyco…. and the resulting “green gap”
+
 
+
TeLuc differes from its deep-see mother ortholog only in 3 Amino Acid changes in Substrate Binding pocket (D19S/D85N/C164H), which basically allows Diphenylterazine (DTZ) to bind.
+
This improved and better part could catalyse a whole new and bright era of characterisation of Synthetic biology.  ___
+
 
+
 
+
 
+
 
+
furimazine patentier Promega, DTZ not
+
 
+
 
+
 
+
 
+
In 2013, Nanoluc were integrated into the iGEM registry as http://parts.igem.org/Part:BBa_K1159001 .
+
This year we choose to go even a step further. We looked into the literature and found a new red-shifted version of Nanoluc, so called teLuc, with even higher brightness against Background. Teluc differ from Nanoluc mainly in its Substrate Binding Pocket and consists of  3 Amino Acids D19S/D85N/C164H. This results in an redshifted  Peak of Luminescence from 460 nm to 502 nm.  
+
This alone could lead to significant more precise and accurate data leading the way to a new era of highly characterized Parts in synthetic Biology and to achieve even greater science.
+
Not even less , if not even graver is the impact of  the redshift.
+
 
+
Originally the redshift is able to reduce the absorption rate of deep-tissue because shorter wavelength are swallowed by the . In Cyanobacteria a similar if even effect is expected. The main compartment of the Photosystem Chlorophyll, as his absprtion maxima is at 440 nm/ 460 nm repectivly for the kind of Chroplyll. Together with the phcobolisoms?, this results in the following spectra for UTEX 2973. .
+
This would also apply for the most plants that dont even have a greater green gap.
+
 
+
Spectra of Chloropyll B and pheophytin b
+
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC61186/
+
 
+
 
+
Using Nanoluc in photosythetis organisms will still lead to luminescse way above the Background, but still signal will be swallwed, even more if high celldesity were measured. This could lead to significant flaws in quantitative analysis of “things”  espesilly comparing diffenrent grwoth stadia and stadia of the cell with differnt chlrorophlyy values.
+
 
+
Chrorophly absorbtion
+
 
+
With our improved teLuc we overcome all these Problems. With less Absorption of Photosynthtic particals and a signifinkant  less autoluminces of DTZ, we can show, that teLUc lead to a significant improvement in the whole scheme of reliable and reproducible Measurement. In Abb.1 we compared the Registry part with a codon optimised Version and see a … increase in Luminescence over Background. Compaed with the
+
This revolution of mesuremnt will lead to a brigth future of trust in Science.  
+
 
+
  
 +
          <p style="text-align: justify; margin-bottom: 1em;">
 +
            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.
 +
          </p>
 +
        </div>
 +
      </article>
 +
    </section>
 +
  </div>
 +
</div>
 
</html>
 
</html>
 
{{Marburg/footer}}
 
{{Marburg/footer}}

Latest revision as of 23:51, 13 December 2019

I M P R O V E


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.