Biomod/2012/TeamSendai/Experiment

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As a result of the experiment, we got that the Shawn annealing was better than annealing with boiled water. And the tube structure made on Shawn annealing was observed by AFM. This result is following.
As a result of the experiment, we got that the Shawn annealing was better than annealing with boiled water. And the tube structure made on Shawn annealing was observed by AFM. This result is following.
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[[Image:Seiseigo0001.png|thumb|left|200px|annealing with boiled water]][[Image:Seiseigo0007 - コピー.png|thumb|none|200px|annealing based on ” A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads”]]
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[[Image:Seiseigo0007 - コピー.png|thumb|center|200px|annealing based on ” A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads”]]
This object whose shape is rectangular object may be the tube because we consider that the shape of the tube is transformed to rectangle when it is crushed by cantilever.
This object whose shape is rectangular object may be the tube because we consider that the shape of the tube is transformed to rectangle when it is crushed by cantilever.
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For the result, the tube annealed by 20h annealing and 40h annealing could been seen as a band when we did electrophoresis but could not been seen when we observed by AFM. On the other hand, when we observed the tube annealed by the condition based on the article proposed by Dr. Shown Douglas [1] by AFM, we could find some structural object. In general, we considered the best condition of annealing is next; the condition of Mg is 8mM and the speed of cooling is based on the article proposed by Dr. Shawn.  
For the result, the tube annealed by 20h annealing and 40h annealing could been seen as a band when we did electrophoresis but could not been seen when we observed by AFM. On the other hand, when we observed the tube annealed by the condition based on the article proposed by Dr. Shown Douglas [1] by AFM, we could find some structural object. In general, we considered the best condition of annealing is next; the condition of Mg is 8mM and the speed of cooling is based on the article proposed by Dr. Shawn.  
Next,we tried to create the tube which can be decorated with cholesterol to connect it with liposome. We call this tube “connect-able tube”.
Next,we tried to create the tube which can be decorated with cholesterol to connect it with liposome. We call this tube “connect-able tube”.
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[[Image:Administrator 2012-10-13 17hr 14min.png|thumb|center|200px]]
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[[Image:imashun8.png|thumb|center|200px]]
When we annealed the connect-able tube and did electrophoresis, the band could be seen at the similar position compared with normal tube. For this, we consider the connect-able tube also may be created and we could find many granular objects when we observed the connect-able tube by AFM.
When we annealed the connect-able tube and did electrophoresis, the band could be seen at the similar position compared with normal tube. For this, we consider the connect-able tube also may be created and we could find many granular objects when we observed the connect-able tube by AFM.

Revision as of 17:00, 27 October 2012

Team Sendai Top


Contents

Gate formation

We made the Gate and observed whether it is successfully made or not by 3 steps: annealing, electrophoresis, and AFM.

Electrophoresis & AFM


We mixed M13mp18 and staples of tube and annealed them. We tried two annealing method. The one is annealing with boiled water, and the other is annealing based on ”A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads”. And we did electrophoresis using this sample. This results is following.


As a result of the experiment, we got that the Shawn annealing was better than annealing with boiled water. And the tube structure made on Shawn annealing was observed by AFM. This result is following.

annealing based on ” A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads”
annealing based on ” A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads”

This object whose shape is rectangular object may be the tube because we consider that the shape of the tube is transformed to rectangle when it is crushed by cantilever. Then, we tried various patterns of annealing condition; the concentration of Mg and the speed of cooling. We tried 20h annealing(80℃ to 70℃→2min/-1℃,70℃ to 30℃→30min/-1℃,30℃ to 4℃→2min/-1℃)and 40h annealing(80℃ to 70℃→2min/-1℃,70℃ to 30℃→60min/-1℃,30℃ to 4℃→2min/-1℃)and the annealing based on the article proposed by Dr. Shawn Douglas [1].

annealing for 20 hours
annealing for 20 hours
annealing for 40 hours
annealing for 40 hours

For the result, the tube annealed by 20h annealing and 40h annealing could been seen as a band when we did electrophoresis but could not been seen when we observed by AFM. On the other hand, when we observed the tube annealed by the condition based on the article proposed by Dr. Shown Douglas [1] by AFM, we could find some structural object. In general, we considered the best condition of annealing is next; the condition of Mg is 8mM and the speed of cooling is based on the article proposed by Dr. Shawn. Next,we tried to create the tube which can be decorated with cholesterol to connect it with liposome. We call this tube “connect-able tube”.

When we annealed the connect-able tube and did electrophoresis, the band could be seen at the similar position compared with normal tube. For this, we consider the connect-able tube also may be created and we could find many granular objects when we observed the connect-able tube by AFM.

[1] A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads (Shawn M. Douglas,* Ido Bachelet,* George M. Church 2012)

スケールバーの表示。これが筒であるという根拠。

Porter functionality

We carried out electrophoresis to see if the target moved and hybridized to Porter of higher bonding energy and if the target was successfully transported through the three Porters.

Electrophoresis

DNA sequences

Transportation of Target by Porter

Protocol

First, we mixed Porter1 and the target for reaction. Second, we added Porter2, expecting that the target would be passed from Porter1 to Porter2. Finally, we added Porter3, expecting that the target would be passed from Porter2 to Porter3.We waited for 15 minutes at each step, letting the target attach to a Porter of higher bonding energy.

The final concentration of samples were follows.
The target DNA: 0.5 μM
Porter 1,2,3: 1.0 μM
Dilution was performed with 1 x TAE Mg2+. The total volume was 5 μM.

Electrophoresis were performed with:
20% acrylamide gel
1×TAE as running buffers
Constant volt 100 V, 3h

Results

fig.1 Lanes are 1: target and Porter1, 2: target and Porter1 and Porter2, 3: target and Porter1 and Porter2 and Porter3
fig.1 Lanes are 1: target and Porter1, 2: target and Porter1 and Porter2, 3: target and Porter1 and Porter2 and Porter3
fig.2 stained samples
fig.2 stained samples

If the target has passed well through Porters, bands of the target hybridizing with Porter1, 2, and 3 should appear. In the gel, bands above each Porter can be seen as the Porter hybridizing with the target.
In the first 15 minutes, Porter1 attached to the target (fig.1: lane 3). After another 15 minutes, the band of Porter1 and Porter2 hybridizing with the target were seen. In the last 15 minutes, the band of Porter1 and Porter2 and Porter3 were seen.
So the result shows that target hybridized with available Porter during the 15 minutes, and then it moved to the Porter that has higher bonding energy during another 15 minutes. Therefore, this indicates that Porters successfully work as “porter” of the target.
We also saw the gel after staining all the samples (fig.2)

Comparison of Porter and Toehold

We saw that Porter catches the target more effectively than the toehold structure by our simulations. In addition, we actually confirmed this idea through experiments.

Protocol

We compared the carrier set of the target as below.

1. Porter1 and Porter2
2. Toehold A and toehold B

First, we mixed the target and Porter1. At the same time, we also mixed the target and toehold A.Second, we added Porter2 to the sample of Porter1 and the target. At the same time, we added toehold B to the sample of toehold B and the target. We waited for 15 minutes at each step, for the sample to react.
The final concentration of samples were follows.
The target DNA: 0.5 μM
Porter 1,2,3: 1.0 μM
Toehold A,B: 1.0 μM
Dilution was performed with 1 x TAE Mg2+. The total volume was 5 μM.

Electrophoresis were performed with:
20% acrylamide gel
1×TAE as running buffers
Constant volt 100 V, 3h

Results

Fig 3. Lanes are 1: Porter1, 2: Porter2, 3: Target, 4: toehold A, 5: toehold B, 6: target and Porter1, 7: target and Porter1 and 2, 8: target and Porter2, 9: target and toehold A, 10: target and toehold A and toehold B, 11: target and toehold B, 12: 20b ladder  The target was passed between porters (lane 7). But the target didn’t attach to toehold A (lane 9). So, the target cannot be delivered by the toehold structure (lane 10)
Fig 3. Lanes are 1: Porter1, 2: Porter2, 3: Target, 4: toehold A, 5: toehold B, 6: target and Porter1, 7: target and Porter1 and 2, 8: target and Porter2, 9: target and toehold A, 10: target and toehold A and toehold B, 11: target and toehold B, 12: 20b ladder The target was passed between porters (lane 7). But the target didn’t attach to toehold A (lane 9). So, the target cannot be delivered by the toehold structure (lane 10)


Similar to Transportation of Target by Porter, Porters and the target, Porter1 and Porter2 attached to the target respectively. Also, the target was passed between Porters (figure3: lane 7).
As for the toehold structure, the target hybridized with toehold B. On the other hand, in the lane of the target and toehold A, the band of the target was still strong (lane 9). So, the target didn’t hybridize with toehold A.
It follows that toehold A cannot catch the target only in 15 minutes, and that the target cannot be delivered by using toehold structures.
Therefore, we concluded that Porter structure, which has some loops, is more effective and efficient than toehold in terms of catching the target.

Cholesterol tag modification

Electrophoresis

Membrane

Making mini-gate

We prepared a preliminary step to that cell gate insert into the liposome. We designed a smaller tube and attempted to insert into liposomes using it.

Similar to the cell gate, we stretched single-stranded DNA of 10 bases that can be modified cholesterol from the side of this tube.

We attached cholesterol to the single-stranded DNA, and  confirmed by electrophoresis.

We expected that these enter into the hydrophobic portion of the liposome. Then, it is likely to that the tube stick to the liposome.

Making liposome

We examined the appropriate composition of the liposome. And we decided the composition; DOPC:DSPE-PEG2000: Fructose = 100:1:1000.

General methods for formation and functional analysis of liposome with DNA

At first, the lipids mixture as follows was prepared.

DOPC 5mM 10μL

DSPE-PEG2000 0.5mM 1μL

Fructose 10mM 50μL

Tube 20μL

Chloroform 70μL

We dry a sample of that composition under argon gas condition.

For liposome formation, 125μL of 1×TAE Mg2+ were added and incubate for 1 h at room temperature.

When indicated, 1 mM Lucifer Yellow fluorescein was added 1/50 fold of total volume (20 μM final). For DNA staining 1/100 fold Hoechst was added after liposome formations. Fluorescence of DNA by Hoechst was observed by a fluorescence microscope. 

As this picture, we observed liposome. So there seems to be some liposome.


Confirming Gate attaches to the membrane

We use fluorescein to confirm that the tube insert into the liposome.

For example, We put Lucifer Yellow fluorescein into big liposome and made a hole using the α- Hemorijin into liposomes. Then, we observed that fluorescein was flowing out from it. Alpha-hemolysin is toxin which makes a hole in the cell and is often used in experiments liposome system.

The figure shows that fluorescein flowing out from a liposome.

Therefore, we are sure that observing fluorescein would be a confirmatory experiment which our tube stuck to the liposome or not.


FigureA1 Vesicles dyed by lucifer yellow.Positive control  This is the image of the vesicle which  putting α-hemolysin. Dotted yellow line is the position of vesicle.And yellow line area is a domain of a graph expressed by "FigureA2'"'
FigureA1 Vesicles dyed by lucifer yellow.Positive control
This is the image of the vesicle which putting α-hemolysin. Dotted yellow line is the position of vesicle.And yellow line area is a domain of a graph expressed by "FigureA2'"'
FigureA2 Gray scale graph  This is the gray scale graph using ImageJ soft.The area of  this graph is expressed by FigureA1's yellow line. Z-axis's gray scale is  separated to 256 gradation.The differences between maximum and minimum of the gray scale are about 0 gradation.
FigureA2 Gray scale graph
This is the gray scale graph using ImageJ soft.The area of this graph is expressed by FigureA1's yellow line. Z-axis's gray scale is separated to 256 gradation.The differences between maximum and minimum of the gray scale are about 0 gradation.



FigureB1 Vesicles dyed by lucifer yellow Negative control  This is the image only vesicles. Yellow line area is a domain of a graph expressed by "FigureB2'"'
FigureB1 Vesicles dyed by lucifer yellow Negative control
This is the image only vesicles. Yellow line area is a domain of a graph expressed by "FigureB2'"'
FigureB2 Gray scale graph  This is the gray scale graph using ImageJ soft.The area of  this graph is expressed by FigureB1's yellow line. Z-axis's gray scale is  separated to 256 gradation.The difference between maximum and minimum of the gray scale is about 10 gradation.
FigureB2 Gray scale graph
This is the gray scale graph using ImageJ soft.The area of this graph is expressed by FigureB1's yellow line. Z-axis's gray scale is separated to 256 gradation.The difference between maximum and minimum of the gray scale is about 10 gradation.


FigureC1 Vesicles dyed by lucifer yellow.  This is the image of the vesicle which  putting the cholesterol modified mini "cylinder".  Yellow line area is a domain of a graph expressed by "FigureC2'"'
FigureC1 Vesicles dyed by lucifer yellow.
This is the image of the vesicle which putting the cholesterol modified mini "cylinder". Yellow line area is a domain of a graph expressed by "FigureC2'"'
FigureC2 Gray scale graph  This is the gray scale graph using ImageJ soft.The area of  this graph is expressed by FigureC1's yellow line. Z-axis's gray scale is  separated to 256 gradation.The difference between maximum and minimum of the gray scale is about 5 gradation.
FigureC2 Gray scale graph
This is the gray scale graph using ImageJ soft.The area of this graph is expressed by FigureC1's yellow line. Z-axis's gray scale is separated to 256 gradation.The difference between maximum and minimum of the gray scale is about 5 gradation.


‘’FigureA1’’ is positive control image because vesicles have many hole by using α-hemolysin. If our mini “Cylinder” modified cholesterol insert to vesicles, fluorescein is similar to this positive control.‘ ’’FigureB1’’ is negative control image existing only vesicle. ’’FigureC1’’ is the result of adding mini “Cylinder” modified cholesterol to condition of existing only vesicle. Gray scale is separated to 256 gradation. (Using ‘ImageJ’ for analysis.) In ‘’FigureB2’’, the difference of between maximum and minimum of the gray scale is about ‘’’10’’’ gradation. But in ‘’FigureC2’’, the difference of between maximum and minimum of the gray scale is about ‘’’5’’’ gradation. These result indicates modified cholesterol ‘’Cylinder’’ can insert to liposome because the gray scale graph of ‘’FigureC2’’ is similar to positive control.


Biacore

Purpose

Confirming whether min-gate with cholesterol-leg sticks in liposome by using Biacore.

What's Biacore?

Biacore is the device that monitors the interaction between the biological molecules using optical phenomena called surface plasmon resonance (SPR) in real time, without the use of labels. To study an interaction, one of the interaction partners is immobilized onto the sensor surface of a Biacore sensor chip. Immobilization occurs by direct coupling to the surface or via a suitable molecule already coupled to the surface. It is necessary to choose the most suitable sensor tip by contents of the kind of molecules to immobilize and the analysis.

We used sensor tip L1. The matrix is that: lipophilic groups are covalently attached to carboxymethylated dextran, making the surface suitable for direct attachment of lipid membrane vesicles such as liposomes. After attachment, the lipid bilayer structure is retained, facilitating the study of interactions involving transmembrane receptors in membrane-like environments.

Immobilization of lipid

1. Set a sensor tip L1 and running buffer on the SPR sensor.

2. Inject 5% Triton X-100 25 μL when the sensor gram becomes steady.

3. Inject lipid suspended in running buffer 100 μL when the sensor gram becomes steady again.

4. Inject 10mM NaOH 5 μL.

5. Inject the lipid 50 μL.

6. Inject 10mM NaOH 5 μL.

7. Repeat 5-6 until the baseline of the sensor gram does not rise.

8. Check the rise in RU levels is 100 RU or under after injection of 100 μg/mL BSA 25 μL.


Running buffer: 10 mM HEPES 150 mM NaCl pH =7.0

BSA: Bovine Serum Albumin

Measurements of samples

Inject sample 10 μL and start measurement. After end of measurement, inject 10 mM NaOH 5 μL. It is necessary to unify measurement time and RU levels when inject sample.

Sample 1:

min-gate (4.2 nM) 50 μL

Cholesterol 12 μL

Running buffer 38 μL

Sample2:

min-gate 50 (4.2 nM) μL

Running buffer 50 μL

Results

From the graph, we confirmed that min-gate with cholesterol-leg had higher RU levels than only min-gate. It shows that min-gate with cholesterol-leg has higher strength of binding. Therefore, we can insert min-gate into liposome by attaching cholesterol-leg.(グラフを貼る)

References

Biacore Life Sciences https://www.biacore.com/lifesciences/index.html

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