Team:Marburg/test joana
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.
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.
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).