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Revision as of 07:50, 27 October 2012
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.
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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 concentration of the target was one-second of that of Porters.
If the target has passed well through Porters, bands of the target hybridizing with Porter1, 2, and 3 should appear from the left side.In the gel, bands are above each Porter.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 Porter (1, 2, and 3) successfully work as “porter” of the target.
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) The gel was 20% acrylamide. The electrophoresis was at 150V for 3hours at 4 degrees. 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. We compared the carrier set of the target as below.1. Porter1 and Porter22. 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. Similar to the interaction of Porters and the target, Porter1 and Porter2 attached to the target respectively (figure2: lane6 and 8). Also, the target was passed between Porters (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 is 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
Confirming interaction of Porters and the target
To see if the target is passed to the porter of higher bonding energy by hybridization, and successfully transported between three porters, we did electrophoresis.
First, we mixed porter 1 and the target for reaction.
Second, we added porter 2, expecting that the target is passed from porter1 to porter2.
Finally, we added porter 3, expecting that the target is passed from porter2 to porter3.
We waited for 15 minutes at each step, for the target attach to a porter of higher bonding energy. The concentration of the target was one-second hat of porters.
When the target is passed well between the porters, from the left, bands of the target hybridizing with Porter1, 2, and 3 should appear.
In the gel, bands are above each Porters.
The result shows that target hybridized with available Porter during 15 minutes, and then it moved to Porter which has higher bonding energy during another 15 minutes. Therefore, this indicates that Porters successfully works as “porter” of the target in the gate.
Comparing effectiveness and efficiency of porter and toehold
We saw that Porters can catch the target more effectively than the toehold structure by the simulation. In addition, we actually confirmed this idea through experiment.
We compared the carrier set of the target as below.
porter1 and porter2
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.
Similar to Interaction of Porters and the target, porter 1 and porter2 attached to the target respectively (figure2: lane6 and 8). Also, the target was passed between porters(lane 7).
As for the toehold structure, the target hybridized with toehold B. On the other hand, in the lane of target and toehold A, the band of the target is still strong (lane 9). So the target didn’t hybridized with toehold A. It follows that toehold A cannot catch the target only for 15 minutes, and that the target cannot be delivered between toehold structures.
Therefore, we concluded that porter structure, which has some loops, is more effective and efficient than toehold when they catch the target.
fig.2 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) 20kb 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 toehold structure(lane 10) The gel was 20 % acrylamide. The electrophoresis was for hours at 4 degrees.
Confirming working of Porter in the gate
To confirm working of "Porters" in the gate, we prepared a gate with and without Porters.
We mixed each gate and the target and made four samples.
They are Target and Gate with P1, Target and Gate with P2, Target and Gate with P1 and P2, and Target and Gate without Porter.
We did electrophoresis of them. The results is following.
We successfully confirmed that Porters can also transport the target in the gate.
Making the Gate
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. This object whose shape is rectangular object may be tube because we consider that the shape of the tube is transformed when it is crushed by cantilever.
Determining the annealing time
Next,we compared the speed of cooling.We tried 20h annealing,(２０hアニーリングの条件を書く)40h annealing,（４０hアニーリングの条件を書く）and the annealing based on the article proposed by Dr. Shown Douglas .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  by AFM, we could find some structual 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.Shown.
Adding cholesterol to the Gate
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 guranular objects when we observed the connect-able tube by AFM.
We purified the annealed Gate.
Because when we do experiment that the target binds porter in the Gate, it is better there are no sufficient staples and porter which is not in the Gate. Our purification is divided into two steps. First, we purified the Gate by using filter(フィルター精製書き方わからない), and do electrophoresis of the Gate. The results is as follows. Next, we cut band of the Gate and purified by using spincolumn(?). By these steps, we succeeded to remove sufficient staples and porter from the 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.
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.
The composition of liposome
DOPC 5mM 10μL
DSPE-PEG2000 0.5mM 1μL
Fructose 10mM 50μL
Chloroform 70μL(90μL:when Tube is not used )
We dry a sample of that composition, and we add
1×2/100mM Lucifer Yellow fluorescein 2.5μL
1×TAE Mg2+ 121.25μL
After that, we observe by a fluorescence microscope.
We examined the appropriate composition of the liposome. And we decided the composition; DOPC:DSPE-PEG2000: Fructose = 100:1:1000. We put this composition of DOPC, DSPE-PEG2000, and Fructose into a glass tube and then, we added chloroform. We dried chloroform in this mixture using argon gas, and we also dried by vacuum equipment. As this picture, we observed liposome. So there seems to be some liposome.