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Team:Tokyo-NoKoGen/Project/phototaxis
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<h2>Introduction</h2> | <h2>Introduction</h2> | ||
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| - | + | There are several parts for the functional addition of chemotaxis to <i>E.coli</i>, but it requires some chemical compounds and its gradient of concentration. When we want to collect <i>E.coli</i> from solution (especially in case large amount of volume), we need to add a large amount of chemical compounds. It is not convenient for the easy collecting.<br><br> | |
There are several reports about the natural bacteria which has phototaxis. Halophilic archaea is one of the major groups of it and well researched bacteria. A few report tried to engineer <i>E.coli</i> to add phototaxis[1][2]. We referred those papers and tried creating a phototaxis device for easy collecting considering about the practical use. | There are several reports about the natural bacteria which has phototaxis. Halophilic archaea is one of the major groups of it and well researched bacteria. A few report tried to engineer <i>E.coli</i> to add phototaxis[1][2]. We referred those papers and tried creating a phototaxis device for easy collecting considering about the practical use. | ||
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Phototaxis device comprises a chimeric fusion protein, NpSRII-9a.a.linker-NpHtrII-StTar (M-fusion). The paper concluded that the fusion chimeric protein was constructed and succeeded in mediating retinal-dependent phototaxis response in <i>E.coli</i>[1].<br> | Phototaxis device comprises a chimeric fusion protein, NpSRII-9a.a.linker-NpHtrII-StTar (M-fusion). The paper concluded that the fusion chimeric protein was constructed and succeeded in mediating retinal-dependent phototaxis response in <i>E.coli</i>[1].<br> | ||
| - | <img src="https://static.igem.org/mediawiki/2010/d/d9/Phototaxix_fig2.png" /> | + | <center><img src="https://static.igem.org/mediawiki/2010/d/d9/Phototaxix_fig2.png" width="50%" /></center> |
NpSRII and NpHtrII are derived from N.pharaonis, and StTar is from <i>Salmonella enteric</i> serovar Typhimutium. NpSRII is retinylidene photoreceptors sensory rhodopsin, and it transmits signals to its transducer protein, NpHtrII. NpHtrII has domains which homologous to chemotaxis transducers of StTar. | NpSRII and NpHtrII are derived from N.pharaonis, and StTar is from <i>Salmonella enteric</i> serovar Typhimutium. NpSRII is retinylidene photoreceptors sensory rhodopsin, and it transmits signals to its transducer protein, NpHtrII. NpHtrII has domains which homologous to chemotaxis transducers of StTar. | ||
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<h2>How does this device work in EcoTanker?</h2> | <h2>How does this device work in EcoTanker?</h2> | ||
When expressed in <i>E.coli</i>, NpSRII is capable of binding of all-trans retinal to form a blue-green-light-absorbing pigment, and NpHtrII mediates the phototaxis response to chemotaxis receptor, StTar. Finally, this response turns off <i>E.coli</i> flagellar motor.<br> | When expressed in <i>E.coli</i>, NpSRII is capable of binding of all-trans retinal to form a blue-green-light-absorbing pigment, and NpHtrII mediates the phototaxis response to chemotaxis receptor, StTar. Finally, this response turns off <i>E.coli</i> flagellar motor.<br> | ||
| - | <img | + | <center><img src="https://static.igem.org/mediawiki/2010/0/0d/Phototaxix_fig3.png" alt="" width="40%" /></center> |
<h2>Progress</h2> | <h2>Progress</h2> | ||
<h3>Construction of parts and devices</h3> | <h3>Construction of parts and devices</h3> | ||
| - | We cloned three entire genes (<i>NpSRII, NpHtrII, StTar</i>) from <i>N.pharaonis</i> and <i>S.typhimurium</i> with primer sets (Primer 1 and 10, 11 and 12, 17 and 6 respectively) which were designed based on 'Standard part fabrication' described in | + | <p>We cloned three entire genes (<i>NpSRII, NpHtrII, StTar</i>) from <i>N.pharaonis</i> and <i>S.typhimurium</i> with primer sets (Primer 1 and 10, 11 and 12, 17 and 6 respectively) which were designed based on 'Standard part fabrication' described in |
(http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication) | (http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication) | ||
These genes were cloned into pSB1C3 and obtained constructs were registered as (NpSRII:BBa_K317000, NpHtrII:BBa_K317001, StTar: BBa_K317002).<br> | These genes were cloned into pSB1C3 and obtained constructs were registered as (NpSRII:BBa_K317000, NpHtrII:BBa_K317001, StTar: BBa_K317002).<br> | ||
| - | <img src="https://static.igem.org/mediawiki/2010/4/45/Phototaxix_fig4.png" alt=""> | + | <center><img src="https://static.igem.org/mediawiki/2010/4/45/Phototaxix_fig4.png" alt="" width="50%"></center></p> |
<br> | <br> | ||
| - | The chimeric fusion | + | <p>The chimeric fusion construct (NpSRII-9a.a.linker-NpHtrII-StTar) was constructed with primer sets (Primer 1~6) by overlap extension PCR method and registered as BBa_K317003.<br> |
| + | <center><img src="https://static.igem.org/mediawiki/2010/3/33/Phototaxix_fig5.png" width="50%" alt="" ></center><br></p> | ||
| - | To demonstrate phototaxis in <i>E.coli</i>, we tried to construct the phototaxis device by adding promoter (BBa_J23111) and rbs (BBa_B0034) on the upstream of NpSRII-9a.a.linker_NpHtrII-StTar (BBa_K317003).<br><br> | + | To demonstrate phototaxis in <i>E.coli</i>, we tried to construct the phototaxis device by adding promoter (BBa_J23111) and rbs (BBa_B0034) on the upstream of NpSRII-9a.a.linker_NpHtrII-StTar (BBa_K317003).<br> |
| + | |||
| + | <center><img src="https://static.igem.org/mediawiki/2010/d/d4/Phototaxix_fig6.png" width="50%" alt="" /></center><br> | ||
All primers used for the construction is shown in below table.<br> | All primers used for the construction is shown in below table.<br> | ||
| - | + | <center><img src="https://static.igem.org/mediawiki/2010/2/28/Phototaxix_fig7.png" alt="" width="95%" /></center> | |
| - | We expressed phototaxis device (BBa_K317028) in <i>E.coli</i> DH5α and evaluated the function. The <i>E.coli</i> was pre-cultured for 1hour in LB medium containing 2 μM all-trans retinal. Then,10 μL of pre-cultured solution was dropped into semisolid medium containing 0.5% agar and 2 μM all-trans retinal, and incubated overnight under light or dark condition. As light source, we used 5 mm Bluish Green LED (OSBG5111A made by OptoSupply Limited) which produces 505 nm wavelength and constant current (16.8 mA) was applied to each LED (3 LEDs / one plate).<br><br> | + | We expressed phototaxis device (BBa_K317028) in <i>E.coli</i> DH5α and evaluated the function. The <i>E.coli</i> was pre-cultured for 1hour in LB medium containing 2 μM all-trans retinal. Then,10 μL of pre-cultured solution was dropped into semisolid medium containing 0.5% agar and 2 μM all-trans retinal, and incubated overnight under light or dark condition. As light source, we used 5 mm Bluish Green LED (OSBG5111A made by OptoSupply Limited) which produces 505 nm wavelength and constant current (16.8 mA) was applied to each LED (3 LEDs / one plate).<br> |
| + | <center><img src="https://static.igem.org/mediawiki/2010/1/1b/Phototaxix_fig8.png" width="65%" alt=""></center><br> | ||
<h3>Result</h3> | <h3>Result</h3> | ||
Colony was more spread out under the dark condition than the light condition. Practically, the average colony area under the dark condition was about twice compared with the colony area under the light condition. As a control experiment, we carried out the same experiment with BBa_K317003 (without promoter and RBS) instead of BBa_317028 (phototaxis device). When <i>E.coli</i> which having phototaxis device senses the 500 nm light, they turns off their flagellar motor. So, this result means that our phototaxis device properly works as we expected !!<br> | Colony was more spread out under the dark condition than the light condition. Practically, the average colony area under the dark condition was about twice compared with the colony area under the light condition. As a control experiment, we carried out the same experiment with BBa_K317003 (without promoter and RBS) instead of BBa_317028 (phototaxis device). When <i>E.coli</i> which having phototaxis device senses the 500 nm light, they turns off their flagellar motor. So, this result means that our phototaxis device properly works as we expected !!<br> | ||
| - | + | <center><img src="https://static.igem.org/mediawiki/2010/d/d6/Phototaxix_fig9.png" width="50%" alt=""><img src="https://static.igem.org/mediawiki/2010/e/e3/Phototaxix_fig10.png" width="50%" alt=""></center> | |
| - | + | ||
<h2>References</h2> | <h2>References</h2> | ||
Latest revision as of 03:03, 28 October 2010
Introduction
There are several parts for the functional addition of chemotaxis to E.coli, but it requires some chemical compounds and its gradient of concentration. When we want to collect E.coli from solution (especially in case large amount of volume), we need to add a large amount of chemical compounds. It is not convenient for the easy collecting.There are several reports about the natural bacteria which has phototaxis. Halophilic archaea is one of the major groups of it and well researched bacteria. A few report tried to engineer E.coli to add phototaxis[1][2]. We referred those papers and tried creating a phototaxis device for easy collecting considering about the practical use.
Why is this device needed?
Phototaxis is used for the system for the EcoTanker guiding. We modified the EcoTanker with the phototaxis, which alters their swimming behavior in response to change in light intensity. After uptaking and storing of chemical compounds into EcoTank, the EcoTankers will be guided by the light signals to a desired location for easy collecting.
What is this device composed inside it?
Phototaxis device comprises a chimeric fusion protein, NpSRII-9a.a.linker-NpHtrII-StTar (M-fusion). The paper concluded that the fusion chimeric protein was constructed and succeeded in mediating retinal-dependent phototaxis response in E.coli[1].
How does this device work in EcoTanker?
When expressed in E.coli, NpSRII is capable of binding of all-trans retinal to form a blue-green-light-absorbing pigment, and NpHtrII mediates the phototaxis response to chemotaxis receptor, StTar. Finally, this response turns off E.coli flagellar motor.
Progress
Construction of parts and devices
We cloned three entire genes (NpSRII, NpHtrII, StTar) from N.pharaonis and S.typhimurium with primer sets (Primer 1 and 10, 11 and 12, 17 and 6 respectively) which were designed based on 'Standard part fabrication' described in
(http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication)
These genes were cloned into pSB1C3 and obtained constructs were registered as (NpSRII:BBa_K317000, NpHtrII:BBa_K317001, StTar: BBa_K317002).
The chimeric fusion construct (NpSRII-9a.a.linker-NpHtrII-StTar) was constructed with primer sets (Primer 1~6) by overlap extension PCR method and registered as BBa_K317003.
To demonstrate phototaxis in E.coli, we tried to construct the phototaxis device by adding promoter (BBa_J23111) and rbs (BBa_B0034) on the upstream of NpSRII-9a.a.linker_NpHtrII-StTar (BBa_K317003).
All primers used for the construction is shown in below table.
Result
Colony was more spread out under the dark condition than the light condition. Practically, the average colony area under the dark condition was about twice compared with the colony area under the light condition. As a control experiment, we carried out the same experiment with BBa_K317003 (without promoter and RBS) instead of BBa_317028 (phototaxis device). When E.coli which having phototaxis device senses the 500 nm light, they turns off their flagellar motor. So, this result means that our phototaxis device properly works as we expected !!
References
[1]Jung et al. (2001) An Archeaeal photosignal-Transducing Module Mediates Phototaxis in Escherichia coli J. Bacteriol. 183, 6365-6371[2]Vishwa D. et al. (2003) photostimulation of a Sensory Rhodopsin ll/Htrll/Tsr Fusion Chimera Activates CheA-Autophosphorylation and CheY-Phosphotransfer in Vitro Biochemistry 42, 13887-13892
