Difference between revisions of "Team:Marburg/test joana"

Line 130: Line 130:
 
                 plant synthetic biology community to accelerate their research.<br>
 
                 plant synthetic biology community to accelerate their research.<br>
 
                 <figure style="text-align:center">
 
                 <figure style="text-align:center">
                        <img style="height: 650px; width: 900px;"
+
                    <img style="height: 650px; width: 900px;" src="https://static.igem.org/mediawiki/2019/1/1c/T--Marburg--Nanoluc.jpg
                            src="https://static.igem.org/mediawiki/2019/1/1c/T--Marburg--Nanoluc.jpg
+
 
                   " alt="Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus UTEX 2973 Absorption spectra">
 
                   " alt="Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus UTEX 2973 Absorption spectra">
                        <figcaption style="max-width: 2400px; text-align: center">
+
                    <figcaption style="max-width: 2400px; text-align: center">
                            Fig.2 - Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus UTEX 2973 Absorption spectra
+
                        Fig.1 - Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus
                        </figcaption>
+
                        elongatus UTEX 2973 Absorption spectra
                    </figure>
+
                    </figcaption>
 
+
                </figure>
 
                 <br>
 
                 <br>
 +
                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.<br>
 +
                To demonstrate the redshift, transformed both NanoLuc <a
 +
                    href="http://parts.igem.org/Part:BBa_K1159001">BBa_K1159001</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.<br>
 +
                <figure style="text-align:center">
 +
                    <img style="height: 650px; width: 900px;" src="https://static.igem.org/mediawiki/2019/e/ee/T--Marburg--Normalized_Luminescence.jpg
 +
                      "
 +
                        alt="Normalized Luminescence measurements of TeLuc and NanoLuc over their full spectra in e.coli.">
 +
                    <figcaption style="max-width: 2400px; text-align: center">
 +
                        Fig.2 - Normalized Luminescence measurements of TeLuc and NanoLuc over their full spectra in
 +
                        e.coli.
 +
                    </figcaption>
 +
                </figure>
 +
                <br>
 +
                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.<br>
 +
                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.<br>
 +
                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.<br>
 +
                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”<br>
  
            <div>
+
                TeLuc differes from its deep-see mother ortholog only in 3 Amino Acid changes in Substrate Binding
                 <p>
+
                 pocket (D19S/D85N/C164H), which basically allows Diphenylterazine (DTZ) to bind.
                    Introduction of exogenous DNA can be done in multiple ways and propagated in a strain if it is
+
                This improved and better part could catalyse a whole new and bright era of characterisation of Synthetic
                    integrated in the
+
                biology<br>
                    chromosome or stably expressed on a self-replicating plasmid.<br> For rapid prototyping in
+
                In 2013, Nanoluc were integrated into the iGEM registry as <a
                    cyanobacteria
+
                    href="http://parts.igem.org/Part:BBa_K1159001">BBa_K1159001</a> .
                    self-replicating plasmids are of higher interest than genome-integrations, as the latter can be
+
                This year we choose to go even a step further. We looked into the literature and found a new red-shifted
                    quite
+
                version of Nanoluc, so called teLuc, with even higher brightness against Background. Teluc differ from
                    time-consuming in cyanobacterial strains with multiple genome copies (<a
+
                Nanoluc mainly in its Substrate Binding Pocket and consists of 3 Amino Acids D19S/D85N/C164H. This
                        href="https://www.ncbi.nlm.nih.gov/pubmed/22092711">Griese <i>et al.,</i> 2011</a>).
+
                results in an redshifted Peak of Luminescence from 460 nm to 502 nm.
                    Furthermore, genes
+
                This alone could lead to significant more precise and accurate data leading the way to a new era of
                    introduced in self-replicating vectors have been shown to have higher gene-expression levels than
+
                highly characterized Parts in synthetic Biology and to achieve even greater science.
                    those integrated
+
                Not even less , if not even graver is the impact of the redshift.<br>
                    in the genome, as copy numbers are typically higher (<a
+
                Originally the redshift is able to reduce the absorption rate of deep-tissue because shorter wavelength
                        href="https://doi.org/10.1099/mic.0.000377">Chen Titel anhand dieser DOI in Citavi-Projekt
+
                are swallowed by the . In Cyanobacteria a similar if even effect is expected. The main compartment of
                        übernehmen <i>et
+
                the Photosystem Chlorophyll, as his absprtion maxima is at 440 nm/ 460 nm repectivly for the kind of
                            al.,</i> 2016</a>) – a desirable trait, not just for rapid prototyping in research
+
                Chroplyll. Together with the phcobolisoms?, this results in the following spectra for UTEX 2973. .
                    applications, but also for
+
                This would also apply for the most plants that dont even have a greater green gap.<br>
                    biotechnological production of valuable compounds.<br>
+
                Using Nanoluc in photosythetis organisms will still lead to luminescse way above the Background, but
                    With our shuttle-vectors encompass a cyanobacterial origin of replication (ori) from
+
                 still signal will be swallwed, even more if high celldesity were measured. This could lead to
                    <i>Synechococcus
+
                significant flaws in quantitative analysis of “things” espesilly comparing diffenrent grwoth stadia and
                        elongatus</i> PCC7942 as well as an <i>E.coli</i> ori, which is perfect for fast cloning
+
                stadia of the cell with differnt chlrorophlyy values.<br>
                    processes, as these
+
                    vectors can be easily recovered from the cyanobacteria and reintroduced in an <i>E.coli</i>
+
                    strain.<br>
+
                    <br>
+
                 </p>
+
 
                 <br>
 
                 <br>
                 <p style="font-size: 20px">
+
                 <b>Chrorophly absorbtion </b>
                    Currently existing shuttle vectors for cyanobacteria are still based on standard systems working
+
                    with multiple
+
                    cloning sites (MCS) for expression of homologous genes (<a
+
                        href="https://doi.org/10.1099/mic.0.000377">Chen Titel anhand dieser DOI in Citavi-Projekt
+
                        übernehmen <i>et
+
                            al.,</i> 2016</a>). A huge downside is that these vectors include either an MCS (e.g.
+
                    pAM5188) or a
+
                    fluorescence reporter (e.g. pAM4787), which is unpractical for easy selection of recombinant clones.
+
                    Additionally,
+
                    an MCS comes with possible sequence constraints due to restriction sites leaving unwanted base pairs
+
                    in your
+
                    constructs.<br>
+
                    Facilitating and standardizing the process of engineering biological systems is one of the
+
                    fundamental goals of
+
                    synthetic biology (<a href="https://doi.org/10.1186/1754-1611-2-5">Shetty Titel anhand dieser DOI in
+
                        Citavi-Projekt übernehmen <i>et al.,</i> 2008</a>), so the
+
                    construction of a shuttle-vector based on a modular cloning method significantly improves the
+
                    genetic toolbox we
+
                    created for genetic engineering and synthetic biology approaches in <i>S.elongatus</i> and other
+
                    cyanobacteria.<br>
+
                </p>
+
 
                 <br>
 
                 <br>
                 <p style="font-size: 20px">
+
                 With our improved teLuc we overcome all these Problems. With less Absorption of Photosynthtic particals
                    The commonly used <i>S.elongatus</i> strain PCC7942 carries two endogenous plasmids, the 46,4kb pANL
+
                and a signifinkant less autoluminces of DTZ, we can show, that teLUc lead to a significant improvement
                    (<a href="https://www.ncbi.nlm.nih.gov/pubmed/18353436">Chen <i>et al.,</i> 2008</a>) which is
+
                in the whole scheme of reliable and reproducible Measurement. In Abb.1 we compared the Registry part
                    essential and the
+
                with a codon optimised Version and see a … increase in Luminescence over Background. Compaed with the
                    7,8kb pANS (<a href="https://www.ncbi.nlm.nih.gov/pubmed/1552863">Van der Plas <i>et al.,</i>
+
                This revolution of mesuremnt will lead to a brigth future of trust in Science.<br>
                        1992</a>) which is
+
                We adopted the redshifted version of NanoLuc, teLuc to the iGEM registry. This allows Teams the use of a
                    not essential for the strain and can easily be cured.<br>
+
                brighter red shifted Luciferase, which peaks at 502 nm. TeLuc differs from NanoLuc in 3 Amino Acid
                    This small plasmid has already been used for construction of shuttle vectors (<a
+
                changes (D19S/D85N/C164H), which leads to high affinity to Diphenylterazine (DTZ).  
                        href="https://doi.org/10.1016/0076-6879(87)53054-3">Kuhlemeier Titel anhand dieser DOI in
+
 
                        Citavi-Projekt übernehmen & van Arkel, 1987</a> ; <a
+
            </p>
                        href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC217787/">Golden & Sherman, 1983</a> ; <a
+
    </div>
                        href="https://doi.org/10.1099/mic.0.000377">Chen Titel anhand dieser DOI in Citavi-Projekt
+
    <br>
                        übernehmen <i>et al.,</i> 2016</a>). <br>
+
    </main>
                    We followed this lead to create the best shuttle-vector available for cyanobacteria by encompassing
+
                    the minimal
+
                    replication region of pANS and the ColE1 origin of replication into our vectors, allowing for stable
+
                    self-replication with high copy numbers in cyanobacteria (<a
+
                        href="https://doi.org/10.1099/mic.0.000377">Chen Titel anhand dieser DOI in Citavi-Projekt
+
                        übernehmen
+
                        <i>et al.,</i> 2016</a>) and <i>E.coli</i> (<a
+
                        href="https://doi.org/10.1016/S0065-2660(02)46013-0">Gerhart Titel anhand dieser DOI in
+
                        Citavi-Projekt übernehmen
+
                        <i>et al.,</i>2002</a>). This addition to the genetic toolbox proves invaluable, as it can be
+
                    easily recovered
+
                    from the cyanobacterial strain and reintroduced in <i>E.coli</i> for fast GoldenGate-based cloning
+
                    processes.<br>
+
                </p>
+
                <br>
+
                <p style="font-size: 20px">
+
                    In order to supply the community with an easy selection system, we equipped our shuttle vector with
+
                    a fluorescent
+
                    reporter that is cut out when introducing new genetic parts:<br>
+
                    A mRFP (red fluorescent protein) cassette is flanked by our standardized TypeIIS restriction enzyme
+
                    recognition
+
                    sequences (BsmBI or BsaI depending on what level you want to clone in). In a standard Golden Gate
+
                    reaction this
+
                    cassette will drop out and leave space for the parts that should be introduced, allowing for easy
+
                    selection on
+
                    plate after successful cloning – red colonies are wrong, still harboring the mRFP cassette and white
+
                    colonies (if
+
                    no other fluorescence is introduced) are correct, as the mRFP was switched with the parts of
+
                    interest.<br>
+
                    <br>
+
                    This crucial part comes in two variations - one for cloning Lvl1 and one for Lvl2 constructs -,
+
                    giving the Golden
+
                    Gate community everything they need for successful and reliable creation of self-replicating vectors
+
                    in
+
                    cyanobacteria.
+
                </p>
+
                <br>
+
                </p>
+
            </div>
+
            <br>
+
            </main>
+
  
 
</html>
 
</html>

Revision as of 01:47, 22 October 2019

I M P R O V E

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. 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 solution: A mutated Version of nanoLuc, so called teLuc
BBa_BBa K3228042 BBa K3228042
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.

Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus UTEX 2973 Absorption spectra
Fig.1 - Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus UTEX 2973 Absorption spectra

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

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
In 2013, Nanoluc were integrated into the iGEM registry as 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.
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.
We adopted the redshifted version of NanoLuc, teLuc to the iGEM registry. This allows Teams the use of a brighter red shifted Luciferase, which peaks at 502 nm. TeLuc differs from NanoLuc in 3 Amino Acid changes (D19S/D85N/C164H), which leads to high affinity to Diphenylterazine (DTZ).