Biomod/2011/TeamJapan/Tokyo/Project/Results: Difference between revisions
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<td align="center"><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results_of_DNA_Ciliate_Body"><img src="http://openwetware.org/images/4/4a/BIOMOD_Tokyo20111031Result_figure_ciliate.png" border=0 width=200 height=200></a></td> | |||
<td><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results_of_Free_Moving_Mode"><img src="http://openwetware.org/images/a/ac/BIOMOD_Tokyo20111031Result_figure1.png" border=0 width=200 height=200></a></td> | <td><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results_of_Free_Moving_Mode"><img src="http://openwetware.org/images/a/ac/BIOMOD_Tokyo20111031Result_figure1.png" border=0 width=200 height=200></a></td> | ||
<td><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results_of_Track_Walking_Mode"><img src="http://openwetware.org/images/0/05/BIOMOD_Tokyo20111031Result_figure2.png" border=0 width=200 height=200></a></td> | <td><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results_of_Track_Walking_Mode"><img src="http://openwetware.org/images/0/05/BIOMOD_Tokyo20111031Result_figure2.png" border=0 width=200 height=200></a></td> |
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<div id="navigation"> <div id="menu" style="position:static"> <ul> <li><a class="aMain" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo">Home</a></li> <li><a class="aTeam" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Team/Students">Team</a></li> <li><a class="aProject" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project">Project</a> <!-- <ul> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project">Overview</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/introduction">Introduction</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Model">Model</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Devices">Devices</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Modes">Modes</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results">Results</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Achievements">Achievements</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Future_works">Future works</a></li> </ul> --> <li><font color="#ffffff">Results</font> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results">Experiments</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Simulations">Simulations</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Achievements/DNA_Devices">DNA Design</a></li> </ul></li> <!-- <li><a class="Simulation" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Simulations">Simulations</a></li> <li><a class="DNA design" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Achievements/DNA_Devices">DNA Designs</a></li> --> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Achievements">Achievements</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Future_works">Future works</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Notebook/Protocols">Protocols</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Notebook/Lab.notebook">Notes</a></li> <li><a class="aNotebook" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Sponsors/">Sponsors</a></li> <li><a class="aSitemap" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Sitemap">Sitemap</a></li>
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Experimental Results
<html><body> <td align="center"><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results_of_DNA_Ciliate_Body"><img src="http://openwetware.org/images/4/4a/BIOMOD_Tokyo20111031Result_figure_ciliate.png" border=0 width=200 height=200></a></td> <td><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results_of_Free_Moving_Mode"><img src="http://openwetware.org/images/a/ac/BIOMOD_Tokyo20111031Result_figure1.png" border=0 width=200 height=200></a></td> <td><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results_of_Track_Walking_Mode"><img src="http://openwetware.org/images/0/05/BIOMOD_Tokyo20111031Result_figure2.png" border=0 width=200 height=200></a></td> <td><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results_of_Light_Irradiated_Gathering_Mode"><img src="http://openwetware.org/images/b/bf/BIOMOD_Tokyo20111031Result_figure3.png" border=0 width=200 height=200></a></td> </body></html>
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The body of the DNA ciliate
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Method
- Two experiments were needed to complete developing DNA ciliate body.
First experiment was creating DNA ciliate by attaching DNAs to polystyrene beads. This process is used the reaction of connecting amino group of aminated DNAs and carboxylic acid of polystyrene beads. We took two Method to react. Both Method are used the common reaction, but chemical materials are different. First method is used EDC and NHS. This induces transforming carboxylic acid to succinimide which is been able to react with aminated DNAs and connect.
- Second method is used EDAC. EDAC reacts with both aminated DNAs and polystyrene beads’ carboxylic acid.
Second experiment is confirming whether deoxyribozyme is attached to polystyrene beads and able to cleave substrate. We confirm deoxyribozyme activity by urea-PAGE. Making mixture of DNA ciliate and substrate and Zn2+ ions. If DNA ciliate has deoxyribozyme activity, substrate is cleaved and the band of cleaved substrate appears as a band.
- Creating DNA ciliate, we use (1) and (2) protocols.
- (1) The method of using EDC and NHS is here.
- (2) The method of using EDAC…
- Confirming DNA ciliate, we use (3) and (4) protocols
- (3) The method of electrophoresis is here.
- (4) The method of making sample is
The results of electrophoresis of DNA ciliate by using EDC
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The results of electrophoresis of DNA ciliate by using EDAC
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In these experimentations, we could confirm two things. First was the deoxyribozyme activity of DNA ciliate body. Second was the firmness of the bond between polystyrene beads and deoxyribozyme. The deoxyribozyme activity of DNA ciliate body can be confirmed. If there is deoxyribozyme activity of DNA ciliate body, the cleaved substrate band is appeared. Furthermore, DNA ciliate body is removed before loading to polyacrylamide gel by centrifuge, so if deoxyribozyme can’t be attached to polystyrene beads firmly, the leg band appeared. In all pictures of gels, there are cleaved substrate bands in lane 8, so it is confirmed that all polystyrene beads are attached deoxyribozyme. Furthermore, there are not deoxyribozyme bands in lane 8, so it is confirmed that all polystyrene beads are attached deoxyribozyme firmly and the deoxyribozyme activity is not because of dislocated deoxyribozyme. In conclusion, we succeeded in making DNA ciliate body. Comparing four results, about both 200 nm and 1um polystyrene beads in diameter, the cleaved substrate band of EDAC method is stronger than EDC method, so DNA ciliate body made by EDAC method is better than EDC method. We decided using DNA ciliate body bodymade by EDAC method.
- Lanes of 5 to 8 are needed for checking deoxyribozyme activity of DNA ciliate body. Lane 5 and 6 are lanes for checking to polystyrene beads. If polystyrene beads had deoxyribozyme activity, the cleaved band would be appeared. Lanes of 7 and 8 are needed for checking that DNA ciliates body have deoxyribozyme activity. If DNA ciliate has normal deoxyribozyme activity, the cleaved band is appeared in lane 8 because metal ions are needed for deoxyribribozyme activity.
Three independent modes of the DNA ciliate
1. Free moving mode
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Method
- To check free moving mode,we used same solution when we checked deoxyribozyme activity The solution is 3% BSA in 1x SSC and the size of DNA ciliate is 200nm (A) and 1um (B). We spotted the solution which DNA ciliates are diffused to glass plate. After that, we put cover glass on it and observed these DNA ciliates by a phase-contrast microscope and took videos.
Results
- The left two videos are used DNA ciliates which are 200 nm in diameter. The right two videos are used DNA ciliates which are 1 um in diameter. The lower videos are enlarged videos of the upper videos. The lower videos are used for taking mote of single DNA ciliate.
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<tr align="center"> <td>[Video](A)</td> <td>[Video](B)</td> </tr> <tr align="center"> <td><iframe width="450" height="259" src="http://www.youtube.com/embed/uGRn9Z8inW4?rel=0" frameborder="0" allowfullscreen></iframe></td> <td><iframe width="450" height="259" src="http://www.youtube.com/embed/-zzB6UeWKoM?hl=ja&fs=1" frameborder="0" allowfullscreen></iframe></td> </tr> <tr align="center"> <td><iframe width="420" height="315" src="http://www.youtube.com/embed/E1vW6eaABcQ" frameborder="0" allowfullscreen></iframe></td> <td><iframe width="420" height="315" src="http://www.youtube.com/embed/jKpgMfls3Kw" frameborder="0" allowfullscreen></iframe></td> </tr>
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- First, we discuss the appropriation of experimentation. To confirm the free moving mode, it was necessary to check the movement of DNA ciliates in solution. By taking videos, we could see the movement of DNA ciliate in solution. Furthermore, the movement is not affected by wind because the solution is isolated from the external environment through cover glass. Because of above two things, this experimentation is appropriate for checking the movement of DNA ciliates.
- Second, we discuss the movement of DNA ciliates in these videos. In both videos, many DNA ciliates moved freely and randomly in the solution. To compare two videos, the DNA ciliates which is 200 nm in diameter moves more strongly than the DNA ciliate which is 1 um in diameter. However some DNA ciliates didn't move because they were crystallization or located at the surface of slide glass or cover glass.
- In conclusion, Brownian motion of DNA ciliates is very intense and at random, so we can say we confirmed free moving mode. Especially, DNA ciliate which is 200 nm moves strongly. By using this mode, DNA ciliate can move on non-DNA glass plate and search other DNAs which can
2. The track walking mode
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Confirmation of Deoxyribozyme activity
- We confirmed deoxyribozyme activity by using electrocataphoresis. This result has already shown in the result page written about DNA ciliate body. (Link:here ) So, we succeeded to confirm deoxyribozyme activity for substrate.
Construction of DNA tracks
Principle and Method of making DNA tracks
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Results of making DNA tracks
- Figure4 is the result of making PDMS mold. We can see two right angle winding lines. They are a part of microchannels and using these microchannels, we arrayed DNAs. The result is Figure5. In Figure5, we hybridized fluorescent labeling complementary DNA strands with arrayed DNA. With the hybridization of arrayed DNAs and fluorescent labeling complementary strands, We can see two fluorescent lines whose shapes are same as the designed microchannel in PDMS mold in Figure5.
- In addition, we made the microchannel which forms like human and hybridized fluorescent labeling complementary DNA strands with arrayed DNA.The result is Figure6.We can confirm that the DNA tracks arrayed as we designed. From the result of Figure5 and 6, we can say that we achieved to array DNA as complex structure and make DNA tracks.
Confirmation of moving directionally
3. Light-irradiated gathering mode
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UV-switching system
- The UV-switching DNA has a stem-loop structure and short blocking DNA, which blocks hybridization of deoxyribozyme. After UV irradiation, this loop becomes open, and hybridize with the deoxyribozyme. (more detail...)
1. Confirmation of UV-switching
Results
- Non-denaturing 20% PAGE result is here.
- U…UV-switching-trap-DNA
- B…Blocking DNA
- D…Deoxyribozyme DNA
- Reaction solution…A 0.225uM and B 0.45uM and D 0.225uM
- All solutions are in 5x SSC (sodium citrate 75mM)
- From left, these bands mean followings.
- U 0.225uM and Mg2+ 80mM
- B 0.45uM and Mg2+ 80mM
- D 0.225uM and Mg2+ 80mM
- U 0.225uM and B 0.45uM and Mg2+ 80mM
- U 0.225uM and D 0.225uM and Mg2+ 80mM (UV isn’t spotted)
- U 0.225uM and D 0.225uM and Mg2+ 80mM (UV is spotted for 60 min.)
- Reaction solution (UV isn’t spotted)
- Reaction solution (UV is spotted for 15 min.)
- Reaction solution (UV is spotted for 60 min.)
- Reaction solution and Mg2+ 80mM (UV isn’t spotted)
- Reaction solution and Mg2+ 80mM (UV is spotted for 15 min.)
- Reaction solution and Mg2+ 80mM (UV is spotted for 60 min.)
- The control bands were appeared in lane 1 to 6. Lane 4 (U 0.225uM and B 0.45uM and Mg2+ 80mM) means the bands when the loop is stable and hybridization U and B (band U-B). Lane 5 (U 0.225uM and D 0.225uM and Mg2+ 80mM (UV isn’t spotted)) means the bands when the loop is open and hybridization of U and D. Lane 6 (U 0.225uM and D 0.225uM and Mg2+ 80mM (UV is spotted for 60 min.)) means the bands when the loop is open and spotted UV (band U-D).
- In the presence of Mg2+, the switching was caused clearly (lane 10 to 12) because of the stable effect of Mg2+.
- Before UV irradiation (lane 10), the UV-switching DNA was closed state (band U-B). After UV irradiation (lane 11, 12), the band shifted to the position of hybridized state (band U-D). Thus, the UV-switching device we designed worked successfully as we intended.
2. Gathering at the specific area
method
- Attaching complementary DNA of deoxyriboazyme on a glass plate
- Making the situation which deoxyribozymes hybridize with complementary DNA on the glass plate
- How to make the situation for hybridization is here
- Putting DNA ciliates on the glass plate
- Waiting for 2 hours
- Observing the DNA ciliates under an fluorescent microscope
result
- A fluorescent image of the DNA ciliates gathering at the spot of complementary DNA is here.
- Complementary DNA was attached on upper-right area in this image.
- There was no DNA in lower-left area in this image.
- DNA ciliates gathered at the spot of complementary DNA, and didn't gather at another area. Following this result, it was confirmed that DNA ciliates can gather at the specific area after UV irradiation.