Biomod/2011/TeamJapan/Tokyo/Notebook/Lab.notebook

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Lab.notebook

Meeting

25.May ・Had a brainstorming.
・Excluded the ideas that clearly unfeasible and categorized rest feasible idea.
13.June ・Narrowed each category of ideas down to a shortlist of projects. Finally, we selected 4 projects.
・By the next meeting, we researched and formed each project
17.June ・Dr.Takinoue gave us lecture of molecular robotics.
21.June ・Adopted controlling micro object by using DNA as our project.
・Discussed the methods of how to control micro object by DNA, and narrowed them down 5 ideas: using light, heat, electric field, magnetic field and tubelin.
・By the next meeting, we researched each of the ideas.
28.June ・Presented the ideas we had researched in detail to each other.
・Considering the presentations, we decided to develop light controlling device. We thought we can use ultraviolet rays, photoligation. or azobenzene as the controlling device.
・As a former step of the controlling, we discussed how to move micro object by DNAs.
29.June ・Came up the idea of moving the object like DNA spider.
・Discussed how attach many DNA to micro object and slide glass as a track of this molecular robot.
5.July ・Discussed the detail of experiments in this summer.
12.July ・Discussed the name of our project.
24.July ・Discussed the name of our project.
・Produced documents for sponsors.
2.August ・Wrote protocols in English.
21.August ・Prepared our presentation for 26 AUG, BIOMOD TeamJapan Midterm Meeting.
22.August ・Prepared our presentation for 26 AUG, BIOMOD TeamJapan Midterm Meeting.
23.August ・Prepared our presentation for 26 AUG, BIOMOD TeamJapan Midterm Meeting.
24.August ・Prepared our presentation for 26 AUG, BIOMOD TeamJapan Midterm Meeting.
25.August ・Prepared our presentation for 26 AUG, BIOMOD TeamJapan Midterm Meeting.
26.August ・BIOMOD TeamJapan Midterm meeting
29.August ・Discussed the plan by Jamboree based on indications from TeamJapan Midterm meeting.
All September,October ・Wrote team wiki, Prepared for presentation, and made youtube video

Developing DNA ciliate body

25.July ・Attached fluoresceinated oligonucleotide DNA probes to 40um glass beads.
3.August ・Attached fluoresceinated DNA probes to 2um polystyrene beads, but the beads was melted.
5.August ・Attached fluoresceinated DNA probes to 2um polystyrene beads, but the beads were clumped.
8.August ・Attached deoxyribozyme DNA probes to 2um polystyrene beads, but the beads was a little clumped.
9.August ・Attached deoxyribozyme DNA proves to 40um glass beads.
・Tested the activity of deoxyribozyme on 40um glass beads by urea-PAGE, but it didn’t worked.
10.August ・Attached deoxyribozyme DNA proves to 1um polystyrene beads. Tried to find the condition of not being clumping.
15.August ・Tested the activity of deoxyribozyme on 1um polystyrene beads by urea-PAGE, but the positive control didn’t work.
18.August ・Attached deoxyribozyme DNA proves to 1um polystyrene beads and 70 um glass beads.
・Tested which buffer deoxyribozyme works in. Decided to use SSC buffer.
19.August ・Tested the activity of deoxyribozyme on 1um polystyrene beads by urea-PAGE, but it didn’t worked.
30.August ・Tried to find the best condition of fixing deoxyribozyme DNA proves to 1um polystyrene beads.
31.August ・Tested the activity of deoxyribozyme on 1um polystyrene beads we made yesterday by urea-PAGE. It worked clearly.
5.September ・Examined the reaction time of attaching deoxyribozyme DNAs to 30um and 1um polystyrene beads.
6.September ・Examined the reaction time of attaching deoxyribozyme DNAs to 30um and 1um polystyrene beads.
7.September ・Tested the activity of deoxyribozyme on 30nm and 1um polystyrene beads we made yesterday by urea-PAGE. The deoxyribozyme on 1um beads didn’t worked. The deoxyribozyme on 30nm worked a little.
12.September ・Examined the reaction time of attaching deoxyribozyme DNAs to polystyrene beads and attached deoxyribozyme DNAs to 200nm polystyrene beads.
13.September ・Examined the reaction time of attaching deoxyribozyme DNAs to polystyrene beads and attached deoxyribozyme DNAs to 1um polystyrene beads.
14.September ・Attached fluoresceinated DNA probes to 70nm polystyrene beads.
15.September ・Tested the activity of deoxyribozyme on 200nm and 1um polystyrene beads by urea-PAGE. The deoxyribozymes of both diameter of beads worked a little.

Developing micrometer-sized tracks

22.July ・Attached fluoresceinated oligonucleotide DNA probes to slide glass in a circle.
25.July ・Processed polyascetal-resin and made a mold of 100um width of micro-channel.
26.July ・Made a PDMS-mold of micro-channel by using polyascetal-mold.
27.July ・Improved the making of polyascetal-mold.
1.August ・Made the brace of PDMS-mold and slide glass.
3.August ・Drained ink on slide glass into PDMS-mold to check the micro-channel.
・Improved the making of the brace of PDMS-mold and slide glass.
5.August ・Checked there were a leak or not when drained silane into PDMS-mold. Because of leaks of silane, we increase the thickness of PDMS-mold to fix the mold and slide glass tightly.
8.August ・Developed 50um width of micro-channel.
9.August ・Tried to find the best condition of draining DNA solution into PDMS-mold.
10.August ・Drained fluoresceinated DNA solution into PDMS-mold Attached on silanized and DSS treated slide glass, but we couldn’t observe fluoresceinated DNA on the glass.
15.August ・Tried to find the best condition of fixing DNA to slide glass.
18.August ・Tried to find the best condition of fixing DNA to slide glass.
19.August ・Tried to find the best condition of fixing DNA to slide glass.
30.August ・Tried to find the best condition of fixing DNA to slide glass.
31.August ・Made various shape of micro-channel.
6.September ・Examined negative controls of attaching DNAs to glass plate.
・Found the results of attaching DNAs to glass plate we did past a month were all nonspecific adsorption.
9.September ・Examined how to remove nonspecific adsorption.
12.September ・Examined how to remove nonspecific adsorption.
13.September ・Examined how to remove nonspecific adsorption.
21.September ・Examined how to remove nonspecific adsorption.
22.September ・Examined how to remove nonspecific adsorption.
26.September ・Arrayed fluorescent labeling DNAs to human form micrometer-sized track.
27.September ・Arrayed substrate of deoxyribozyme to glass plate.
30.September ・Examined the condition of arraying DNAs to glass plate.

Developing DNA devices

22.July ・Designed DNA sequence of deoxyribozyme of DNA ciliate.
27.July ・Checked the detection limit of the concentration of DNA solution by urea-PAGE stained with ethidium bromide.
1.August ・Checked the detection limit of the concentration of DNA solution by urea-PAGE stained with SYBR gold.
5.August ・Tested the activity of deoxyribozyme we designed as deocyribozyme of DNA ciliate, but it didn’t work.
8.August ・Adjusted pH of deoxyribozyme solution buffer and retested the activity of deoxyribozyme, but it worked without divalent metal ions.

Confirming the free moving mode

14.October ・Observed Brownian motion of 200nm and 1um diameter DNA ciliate.

Confirming the track walking mode

All month of September and October Simulated and analyzed the motion of DNA ciliate on DNA track.
27.September ・Examined the condition of hybridizing DNA ciliate with substrate on track.
30.September ・Examined the condition of hybridizing DNA ciliate with substrate on track.
3.October ・Examined the condition of hybridizing DNA ciliate with substrate on track.
5.October ・Observed whether DNA ciliates on substrate-track.
14.Octorber ・Observed whether DNA ciliates on substrate-track.

Confirmation the light-irradiated gathering mode

27.September ・Attached complementary DNA strands(not substrate) of deoxyribozyme at a spot on glass plate.
30.September ・Observed whether DNA ciliates gather at the spot where we attached complementary strands of deoxyribozyme, but they didn’t.
3.October ・Examined the condition of hybridizing DNA ciliate with substrate on track.
・Observed whether DNA ciliates gather at the spot where we attached complementary strands of deoxyribozyme. They gathered at the spot clearly.


Brainstorming

We wanted to move and control the giant object by using nanometer-sized DNA, so it's necessary that we come up with the model to control the giant object.
At this chapter, we expressed the process of deciding model of controlling. When we looked for the model, we came up with an idea to move giant object from ciliate. We thought if we immobilized a lot of single strand DNAs around the beads, we could control the giant-beads by using interaction with the complementary strand. But we also needed to think about the method to control the direction of the bead.
Then, we discussed and enumerated the models as far as we could come up with. Listed models are as follows.
<Control by using--->
Radio wave
Ultraviolet ray
Infrared ray
Azobenzene
Then, we investigated each listed themes. The result of investigation is following.
<Radio wave>
We found the following thesis:Kimberly Hamad-Schifferl et al:, Nature,415(10),152-155(January. 2002). This thesis reports the possibility of controlling the DNA hybridization by using gold atom and radio wave. Then we thought we might control by using this thesis. Our idea was to control the direction of moving beads by irradiating radio wave only one side of beads.
<Ultraviolet ray>
We found the following thesis: Kenzo Fujimoto et al:,Bioorganic & Medicinal Chemistry Letters, 15, 1299-1301(2005). This thesis reports the possibility that we can control photocrosslinking of DNA by using p-carbamoylvinyl phenol nucleoside. We thought if we apply this result, we can control the movement of beads by irradirating ultraviolet ray on one side.
<Infrared ray>
We found the following thesis: Kenji Yasuda et el:,BioTechinques, 28(5), 1006-1011 (May. 2000). This reports the realization of focal extraction of DNA by using photo-thermal denaturation. We thought we could apply to move beads using this report.
For the reason given above, we decided to adopt the proposal of model using three wavelengths.
In the course of our discussion, an idea came into our mind. This idea was to control the direction by using three different wavelengths; radio wave, ultraviolet ray and infrared rays. This model applies to the three thesises above. (About radio wave and Ultraviolet ray and infrared ray) We call this model “Twister-Model”.
<Azobenzene>
We also paid attention to the research of Asanuma who is the professor of JAIST (Japan Advanced Institute of Science and Technology). He found the way to control DNA duplex formation and dissociation by making use of azobenzene. We thought his research could apply to make stop arrangement. Stop arrangement is one of the reversible modes of controlling beads. We assumed that if we crossed DNA tracks and DNA added azobenzene, we would make it possible to make reversible switches which had start and goal points.


However, the single strand DNA added one azobenzene was about 130 dollar. Moreover, as far as we could think, we needed to the DNA added 13 azobenzene. This was too expensive to buy, so we decided to change arraignment and bought DNA added two azobenzene:UV-switching-DNA. The result of changing arraignment, (write benefit) we could not control DNA duplex dissociation, so our model of azobenzene was an irreversible reaction. We used this DNA as the way to gathering.
<Our Model>
The result of our discussion, we decided two models to control beads’ movement. Two models are the control by using three different waves and the control by using azobenzene.
To achieve these models, we established 6 subjects to solve. Six subjects are the following.
  • Experimentation which confirms the moving of beads by using the system used DNA spider. (DNA spider comes from a thesis: Kyle Lund et al:,Nature,vol465,206-210 (May. 2010).)
  • Experimentation which apply the system of DNA walker. (DNA walker comes from a thesis: Hongzhou Gu et al:,Nature,vol465, 202-205(May. 2010).)
  • Modeling of twister-model of a two-dimensional twister-model.
  • Experimentation of a two-dimensional twister-model which uses the system of DNA walker.
  • Experimentation which confirms the movement of DNA tracks when we use the model of three different waves.
  • Experimentation of two-dimensional twister-model which uses the model of three different waves.
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