Biomod/2012/Titech/Nano-Jugglers/Results
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} </style> </head> <BODY> <div id="biomodlink"> <<a href="http://openwetware.org/wiki/Biomod">BIOMOD</a>|<a href="http://openwetware.org/wiki/Biomod/2012">2012</a>|Titech Nano-Jugglers </div> <div id="header"> <div id="navigation"> <div id="menu"> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers"><br>Home<br><br></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Team/Students"><br>Team<br><br></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Project"><br>Project<br><br></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Results">Results<br>&<br>Methods</a></font></li> <li class="ach"><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Achievements"><br>Achievements<br><br></a> <li class="sup"><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Protocols"><br>Suppl. Info.<br><br></a></li> <li class="none"><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Acknowledgement"><br>Acknowledgements<br><br></a></li> </ul> </div> </div> </div> </BODY> </html>
Results
<html><body><td align="center" width="300px"><A href=#0._Construction_of_Biomolecular_Rocket title="Body"><img src="http://openwetware.org/images/b/b5/BM.jpg" border=0 width=310 height=240></a></td></body></html> |
Construction of Biomolecular Rocket
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<html><body><A href=#1._Power_supply_for_the_rail-free_movement title="Rail-free"><img src="http://openwetware.org/images/7/7f/Rail-free%E3%80%80movement_kinesin.jpg" border=0 width=310 height=220></a></body></html> Power supply for the rail-free movement |
<html><body><A href=#2._Increasing_driving_force_for_the_high-speed_movement title="High-speed"><img src="http://openwetware.org/images/8/89/High-speed_movement.jpg" border=0 width=310 height=220></a></body></html> Increasing driving force for the high-speed movement |
<html><body><A href=#3._Introduction_of_a_photo-switchable_DNA_system_for_the_directional_control title="Control"><img src="http://openwetware.org/images/d/dd/Control_image.jpg" border=0 width=310 height=220></a></body></html> Introduction of a photo-switchable DNA system for the directional control |
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0. Construction of Biomolecular Rocket
<html><body><font size="5">We constructed Biomolecular Rocket with a microbead, catalysts, and designed DNAs.</font></body></html> Biomolecular Rocket is composed of a micrometer-sized body and many catalytic engines. The body consists of a microbead with a diameter of 10 μm, and catalytic engines consist of platinum nanoparticles or catalase molecules. The catalytic engines are conjugated to the body using a DNA-based linker in a spatially selective manner.
>>see more methods of construction of Biomolecular Rocket |
<html><body><td align="center"><img src="http://openwetware.org/images/4/4d/Charts.jpg" border=0 width=200 height=440></a></td></body></html> |
0.1. Selective coating of the body
<html><body><font size="5">We succeeded in selective coating of a micrometer-sized bead by vapor deposition of Au and Cr.</font></body></html>- >>see more methods
<html><body><td align="center"><img src="http://openwetware.org/images/a/a5/Vapor_deposition.jpg" border=0 width=330 height=110></a></td></body></html> - >>see more methods
- Figure 0.1a, b and c are microscope images of 40 μm microbeads. Figure 0.1a shows microbeads before vapor deposition of metals. Figure 0.1b shows microbeads after vapor deposition of Au on the microbeads. Figure 0.1c shows microbeads after additional vapor deposition of Cr on the Au-deposited microbeads. The microbeads had three types of surface areas because the angular alignment of the beads was changed when Cr was deposited on the Au-deposited microbeads.
- Similarly, Figure 0.1e, f and g are microscope images of 10 μm microbeads. Figure 0.1e shows microbeads before vapor deposition of metals. Figure 0.1f shows microbeads after vapor deposition of Au on the microbeads. Figure 0.1g shows microbeads after additional vapor deposition of Cr on the Au-deposited microbeads. The 10 μm microbeads probably had three types of surface areas. From these result, we conclude that selective coating of microbeads for Biomolecular Rocket.
0.2. Design of linker DNA strands
- <html><body><font size="5">We designed linker DNA strands that can stably hybridize.</font></body></html>
- >>see more methods
We designed 2 types of DNA duplex that can hybridize stably by NUPACK. We called the longer one as DNA sequence L, and the shorter one as DNA sequence S. We called their complementary strands as DNA sequence L* and DNA sequence S*. |
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- In both Figure 0.2a and 0.2b, Tm is far from Rt. So, we achieved DNA design that can hybridize stably.
0.3. DNA conjugation
0.3.1. Selective conjugation of DNA to polystyrene surface area
<html><body><td align="center"><img src="http://openwetware.org/images/f/f5/Polystyrene_EDAC.jpg" border=0 width=300 height=240></a></td></body></html> <html><body><font size="5">We succeeded in DNA conjugation to polystyrene surface area by using EDAC.</font></body></html>
- >>see more methods
For visualizing the results of DNA conjugation to polystyrene surface area, we hybridized fluorescent complementary DNA and observed them under blue light by microscope.
- >>see more methods
- Figure 0.3.1a, b, and c are images of 10 μm selective coated beads under visible light. Figure 0.3.1a', b', and c' are images of 10 μm selective coated beads under blue light. Figure 0.3.1a anda' shows selective coated beads after conjugated DNA to polystyrene area and hybridize fluorescent cDNA. Figure 0.3.1b and b' shows selective coated beads after conjugated DNA to polystyrene area. Figure 0.3.1c and c' shows selective coated beads after mixing fluorescent DNA. Selective coated beads exhibit fluorescence because fluorescent cDNA was excited by blue light. From these results, we conclude that selective conjugation of DNA to polystyrene surface area.
0.3.2. Selective conjugation of DNA to metal surface area
<html><body><td align="center" width="150px"><img src="http://openwetware.org/images/8/87/Metal_SAM.jpg" border=0 width=300 height=240></a></td></body></html> <html><body><font size="5">We succeeded in DNA conjugation onto Au surface area by the reaction of SAM.</font></body></html>
- >>see more methods
For visualizing the results of DNA conjugation to metal surface area, we hybridized linker DNA to Au plate surface.
- >>see more methods
- Figure 0.3.2a is a images of Au plate that linker DNA conjugated partially. Figure 0.3.2b is a images after soaking up DNA buffer of Au plate. Figure 0.3.2a is a images after washing away of partially DNA conjugated Au plate. Final concentration of phosphate and NaCl buffer are the same in these spot. But 1, 2 and 3 are added NaCl concentration immediately after 24 hours of incubation, the other hand 4, 5, and 6 are added NaCl as salt aging process. Au plate reveals partially hydrophilic, this is because physical property of Au surface was assimilated to physical property of DNA. From these result, we conclude that selective conjugation of DNA to metal surface area.
0.4. Catalyst attachment with DNA hybridization
<html><body><td align="center"><img src="http://openwetware.org/images/1/19/Conjugation_catalyst.jpg" border=0 width=300 height=230></a></td></body></html> <html><body><font size="5">We tried to attach catalyst to the body of Biomolecular Rocket with DNA hybridization.</font></body></html>
DNA hybridization enable us to attach each materials in that DNA strand transits to take thermodynamically stable forms.
- >>see more methods
- >>see more methods
- To verificate this experiments, we tried to attach Pt particles to Au plate (instead of Au deposited beads) , but there is a few difference between DNA conjugated materials and natural materials. So, We must prove the technology of attaching catalyst. DNA strands are nano-scale, and to conjugate micro-scale beads and platinum engines was difficult.
- We also design DNA strands of DNA sequence L and DNA sequence S that have the first 15 bases from 5' end as a linker (TTTTTTTTTTTTTTT). This DNA was also designed not to make unexpected structures. Probably, this linker part will allow much leeway for attaching the body, in that decrease the effect of steric hindrance.
Poly T linked DNA sequence L 5’-TTTTTTTTTTTTTTTCGTCTATTGCTTGTCACTTCCCC-3' Poly T linked DNA sequence S 5’-TTTTTTTTTTTTTTTAATACCCAGCC-3’
1. Power supply for the rail-free movement
<html><body><font size="5">We achieved the power supply for the rail-free movement.</font></body></html>
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<html><body><td align="center"><img src="http://openwetware.org/images/b/b3/WikiRFBM.jpg" border=0 width=240 height=240></a></td></body></html> |
1.1. DNA hybridization in solution of H2O2
- <html><body><font size="5">We succeeded in DNA hybridization in 1%-5% H<sub>2</sub>O<sub>2</sub> solution stably.</font></body></html>
- To visualize the stability of DNA duplex in 1%-5% H2O2 solution, we use PAGE electrophoresis. It shows the difference of molecular weight of nucleic acid that comes from denaturetion or hybridization in the form of bands.
- >>see more methods
- Figure 1.1 is a image of DNA hybridization after immersing in solution of H2O2. Lane 1, 2, 3, and 8 shows dsDNA, that is hybridized after immersing in solution of 0%-5% H2O2 for 90 minutes. Lane 4, 5, 6, and 7 shows ssDNA, after immersing in solution of 0%-5% H2O2 for 90 minutes. Influence of H2O2 solution within 90 minutes was few for DNA hybridizing, because the line of dsDNA appear in the same positions. Also influence of H2O2 solution within 90 minutes was few for ssDNA in that the line of ssDNA appear in the same positions. From this results, we achieved DNA hybridization in solution of H2O2.
1.2. Power supply for rail-free movement by using Pt catalytic engine
<html><body><td align="center" width="150px"><img src="http://openwetware.org/images/8/89/Simple_beads.jpg" border=0 width=240 height=180></a></td></body></html> <html><body><font size="5">We tried to verify whether platinum hemisphere moves forward in solution of H<sub>2</sub>O<sub>2</sub>.</font></body></html>
We provide 1 μm platinum particles, and Cr coating to create platinum hemisphere. Then added 3% H2O2 solution and observed their movement.- >>see more methods
1.3. Power supply for rail-free movement by using catalase catalytic engine
<html><body><td><img src="http://openwetware.org/images/1/1b/Crf.jpg" border=0 width=240 height=180></a></td></body></html> | <html><body><font size="5">We scceeded in rail-free movement by taking advantage of catalase catalytic ability.</font></body></html> Catalase has Catalytic ability of decomposing H2O2, like platinum. We conjugated catalase to the polystyrene hemisperical area, so this catalase hemispherical bead moves by emission of O2 bubbles without rails. |
- Figure 1.3 reveals the movement of 10 μm catalase hemisphere movement in solution of 3% H2O2. We can easily distinguish catalase conjugated bead or natural bead in that their movement in solution of 3% H2O2 is very different. Natural beads didn't move at all, on the other hand catalase hemisphere emitted bubbles and moved quickly. From this results, we conclude that Verification of catalase hemisphere behavior in solution of H2O2.
2. Increasing driving force for the high-speed movement
<html><body><font size="5">Biomolecular Rocket achieved high-speed movement by increasing catalytic surface area.</font></body></html>
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<html><body><img src="http://openwetware.org/images/2/26/Hst.jpg" width=300 height=230></body></html> |
2.1. Analyses of the speed of platinum in solution of H2O2 by High-speed camera
- We succeeded in analyses of the speed of plutinum in solution of H2O2 by High-speed camera.
- >>see more methods
a
<html><iframe width="440" height="330" src="http://www.youtube.com/embed/7pBw4FWEt3I" frameborder="0" allowfullscreen></iframe></html>
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<html><iframe width="440" height="330" src="http://www.youtube.com/embed/E1rtI0mS5Zs" frameborder="0" allowfullscreen></iframe></html>
Fig. 2.1 Analyses of the speed of platinum in solution of H2O2.
a Platinum movement in solution of H2O2 b Analises of the speed of plutinum by High-speed camera
- Figure 2.1b disclosed the values of Acceleration, Velocity, Coordinate x, and Coordinate y of platinum movement. Not only that, by observation of these values, we could determine relationships between bubble radius growth and the speed of platinum, so we were able to simulate of movement of our rocket.
2-2.Simulation for speeding-up of Biomolecular Rocket movement
- From our numerical simurations of high-speed.We carried out numerical simulations of the high-speed movement of Biomolecular Rockets.
- <html><body><td align="center" width="150px"><img src="http://openwetware.org/images/f/f0/Simulation_speed.jpg" border=0 width=240 height=180></a></td></body></html>
3. Introduction of a photo-switchable DNA system for the directional control
Direction of the rail-free movement of Biomolecular Rocket can be controlled, since we introduced a photo-switchable DNA system.
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<html><body><td align="center" width="150px"><img src="http://openwetware.org/images/8/8a/DC.jpg" border=0 width=300 height=230></a></td></body></html> |
3.1. Design of photoresponsive DNA
<html><body><td align="center" width="150px"><img src="http://openwetware.org/images/4/45/Photo_design.jpg" border=0 width=210 height=210></a></td></body></html> <html><body><font size="5">We designed photoresponsive DNA strands in order to control the direction of Biomolecular Rocket.</font></body></html>
In this project, we ensured that photoresponsive DNA could hybridize their cDNA at room temperature by Abs. Numerical values of Abs depends on the concentration of material corresponding to the absorption wavelength.
>>see more methods
- We called DNA as A:Photoresponsive DNA sequence S, and B:Photoresponsive DNA sequence S*. Figure 3.1 reveals that Abs of A+B around 260nm was below those of A and B, and also Abs of A+B around 330 nm was less than that of A. This is because the concentration of azobenzene decreased by hybridization of A with B.
- Calculation an average absorbance of A and B Abs around 260 nm, theorical value of A+B was 0.168. Compared to this, measured value of absorbance is 22.6% lower. We thought this difference comes from that DNA formed duplex and interactions between base pairs, so decrease the UV absorbance relative to single strands.
- In this point, we believe that the complementary photo-responsive DNAs can form duplex. So, we concluded that these results mean A and B hybridized successfully.
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Fig.3.1 Abs of photoresponsive DNA results, wave length around 260 nm reveals the concentration of DNA. Around 330 nm reveals the concentration of trans-formed azobenzene. |
3.2. Dissociation of photoresponsive DNA by UV-light irradiation
<html><body><td align="center" width="150px"><img src="http://openwetware.org/images/f/f3/Azobenzene_image_dissociation.jpg" border=0 width=240 height=180></a></td></body></html> We have scceeded in dissociation of photo-switchable DNA by UV-light irradiation.
Photo-switchable DNA duplex can easily dissociate its duplex by irradiating UV-light. We put this switching system in the rocket in order to control our rocket. To concirm the dissociation of photo-switchable DNA duplex, we checked Abs.- >>see more methods
- To investigate the relationship between the strength of UV light and the time of dissociation to determine the valid time, we examine 2 type of UV light.
- Fig.3-2.1 represents spctrum of Abs in condition of UV-light(30 mW/cm2) irradiation. Abs of A+B around 260 nm was increasing gradually from 0 minutes to 5 minutes. This result means dsDNA was completely dissociated after irradiating UV-light for 5 minutes. Moreover, Abs of A+B around 330 nm was decreasing from 0 minutes to 5 minutes. This means trans-formed azobenzene changed its form to cis-formation. Therefore, we achieved photoresponsive DNA which was designed by us would be dissociated by irradiating UV-light for 5 minutes.
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Fig.3.2.1 Spectrum analysis of photoresponsive DNA duplex(A+B) in condition of UV-light(30 mW/cm2) irradiation |
- Then, we tested the dissociation of photoresponsive DNA under the condition of different strength of UV-light(180 mW/cm2).
- nbsp; Fig.3-2.2 shows photoresponsive dsDNA was dissociated completely after the irradiation of UV-light for 50 seconds. There are three evidences.
- First, from Fig.b), we could find that dsDNA was dissociated gradually from 0 seconds to 50 seconds because maximum Abs around 260 nm was increasing. Second, Fig.c) shows that trans-formed azobenzene decreased because Abs around 330 nm was decreasing from irradiation for 0 seconds to 50 seconds. Finally, Fig.d) shows cis-formed azobenzene increased. As we did experiences for many times, we noticed that there was maximum wave length around 480 nm. By researching, we reached the fact that Abs around 480 nm shows the existence of cis-formed azobenzene. So, we can say that cis-formed azobenzene increased because Abs around 480 nm was increasing from irradiation for 0 seconds to 50 seconds.
- So, we concluded that photo-seichable DNA system achieved after 50 seconds irradiation of UV-light(180 mW/cm2).
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Fig.3.2.2 Spectrum analysis of photoresponsive DNA duplex(A+B) in condition of UV-light(180 mW/cm2) irradiation |
3-3.Simulation for directional control of Biomolecular Rocket
- sss
- If you want to see all of our methods, click here