Biomod/2012/TeamSendai/Experiment: Difference between revisions

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Revision as of 14:26, 26 October 2012

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<body> <div id="Container"> <!-- Menu --> <ul id="menu"> <li><a href="http://openwetware.org/wiki/Biomod/2012/Tohoku/Team_Sendai ">Home</a></li> <li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Idea ">Project</a></li> <li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Design">Design</a> </li> <li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Simulation">Simulation</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Experiment ">Experiment</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Future Application">Future Application</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Diary">Diary</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Team ">Team</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/Tohoku/Team Sendai/header"></a> </li>

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Flow chart

<img src="http://openwetware.org/images/4/4b/Flowchartaota.png" alt="porter" width="400" height="300">



Porter

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. >>DNA sequences 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. 　 >>Click for more detail about this protocol 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


this is Electrophpresis
The Wikipede edits Electrophoresis.


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. 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. 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 Porter works as “porters” in the gate, we prepared a gate with and without porters. We mixed these gates and the target, and did electrophoresis. >>Click for more detail about this protocol We successfully confirmed that Porters can also transport the target in the gate.


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 [1].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 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.

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.


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 Tube 20μL Chloroform 70μL(90μL:when Tube is not used )

We dry a sample of that composition, and we add

1×Hoechst 1.25μL 1×2/100mM Lucifer Yellow fluorescein 2.5μL 1×TAE　Mg2+　121.25μL

After that, we observe by a fluorescence microscope.

Making liposome

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.


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