Biomod/2013/Sendai/experiment

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            <a href="http://openwetware.org/wiki/Biomod/2013/Sendai"><h1 style="color:white;" ><b>Biomod<span>2013<br>&emsp; Team</span>Sendai</b></h1></a>

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<p class="sukima">Experiment </p>

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<article data-title="Experiment">
           <h2>Experiment</h2>

<p> <h3>About</h3></br>

<a href="#experimentsubproject1">内側からアルギン酸膜を破壊するサブプロジェクト</a><br> <a href="#experimentsubproject2">外側からリポソームを破壊するサブプロジェクト <font size="2">リポソーム班</font> </a><br> <a href="#experimentsubproject3">外側からリポソームを破壊するサブプロジェクト <font size="2">B-Z班</font> </a><br>

    </article>

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     <article data-title="Egg type molecular robbot">

<h3>Step1 Egg-type trigger</h3>

<h4>ⅠExperiment list</h4> The experiment necessary for realization of Egg-type trigger is following</br> 1-1) Making liposome in alginate hydro gel beads</br> 1-2 ) preparing of alginate hydro gel membrane containig buffer</br> 2) Function confirmation of PNIPAM</br> 3) Measurement of density of EGTA and time necessary to dissolve alginc acid gel</br> 4) Urea diluting annealing</br></br>

<h4>1-1) Making liposome in alginate hydro gel beads</h4> <h5>Purpose</h5> We need to put liposome in alginate hydrogel beads for chelating agent which in the liposome destroy alginate hydrogel membrane after liposome is broken. at first We make liposome and put it in a sodium alginate water solution and make alginate hydrogel beads with the solution

<h5>Method</h5> We made liposome by passing droplet which covered one fold of phosphatide films in one fold of phosphatide film which was formed in the interface of two things not mixing well like oil and water We made outer buffer by adding oil 70μℓ to glucose 100μℓ.Then, we made inner buffer by adding oil 40µℓ to BSG(fluorescent substance) 1µℓ,and mixed it until it is muddy white by pipetting and tapping. We poured the inner buffer on outer buffer and centrifuged it for 70 seconds.After that, took out the liposome which collected at the bottom of the tube. Then, we added the liposome to 1.5%sodium alginate solution, and we put it in a capillary and dropped it in 0.4M CaCl2 aq by centrifuging it in a device such as Fig1 for 2-3 minutes

Fig1 Experimental device of alginic acid gel beads</br></br>

<h5>Result</h5> Because fluorescence protein is in the liposome, liposome shines. We observed the alginate hydrogel beads by cofocus laser microscope. (Fig 2) because the globe which glittered in alginate hydrogel beads is confirmed, it showed that liposome was in the alginate hydrogel beads.</br>

<img src="http://openwetware.org/images/1/1c/Exp-1-1-00.png" width="400"></br></br> Fig2 Cofocus laser microscope image of alginic acid gel beads with liposome</br></br>

<h5>Discussion</h5> Fig2を見るとリポソームが非常に小さくなってしまっている。これは浸透圧の影響を受けてリポソーム内の水分子が外部に漏れてしまっているためだと考えられる。これはアルギン酸水溶液の濃度やリポソーム内の溶液を調整することで改善できると考えれる。</br> From Fig2,　liposome became very small. It was thought that this is because H2O molecule in the liposome leak under the influence of osmotic pressure outside. This problem may be improved by adjusting the density of sodium alginate solution and solution in the liposome.</br>

</br> <h4>1-2 ) preparing of alginate hydro gel membrane containing buffer<h4> <h5>Purpose</h5> Viscosity of beads made from alginate hydrogel is so thick that DNA can't be annealed because appulse numbers is decreased. So, we have to make alginate hydrogel membrane containing buffer. To make alginate hydrogel membrane containing buffer, we made double nosepiece and did following experiments. </br>

<h5>Principle</h5> When we provided enhanced gravity to double capillary by a centrifuge rotor, enclosed content fluid and extra solution (sodium alginate) effuse from front edge of nosepiece in the state of granularities, content fluid wrapped in sodium alginate. These granularities fall in drops to the solution of sodium alginate and only surface turn into gel and we got membranal gel beads.</br>

<h5>Method</h5> We used double capillary(Fig2), made from outer thick one and inner fine one. To confirm solution in the gel beads, we used 1.5% sodium alginate solution as outer solution, fluorescent stuff and 0.4M CaCl2 aq as inner one. Finally, we put capillary containing solution into centrifuge rotor for a few minutes and fell in drops to 0.4M CaCl2 aq.</br>

    Fig3 二重ノズルの構造</br></br>

<h5>Result</h5> We observed alginate hydrogel by confocal laser microscope and we could confirm fluorescence in the alginate hydrogel. </br>

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  <img src="http://openwetware.org/images/0/07/Exp-1-2-00.png" width="400">
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  <img src="http://openwetware.org/images/8/88/Exp-1-2-01.png" width="400"> 
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<h5>Discussion</h5> Fig4を見てわかるように球体にならずカエルの卵状のゲルができた。これは遠心の速度を変えることで改善できると考えられる。</br> また今回、内管溶液に0.4M CaCl2 aqを使用しているためDNAが凝縮してしまう。これを改善するために濃度の低いMgCl2 aq などを使用する必要がある。 Alginate hydrogel did not become the globe, tube-formed gel was made(like frog spawn) It is thought that this problem can be improved by changing centrifugal speed. </br> In addition, this time, DNA will aggregate because there is Ca2+ in inner solution. It is necessary to use low density MgCl2 aq to improve this problem.</br>

<h4>2) Function confirmation of PNIPAM</h4> <h5>Purpose</h5> In this project, it is necessary that liposome destroys surely when we increase temperature to 32℃.So, we confirmed whether liposome with PNIPAM destroys when the temperature become 32℃.</br>

<h5>Principle</h5> NIPAM is hydrophilic at less than 32℃, but it become hydrophobic and shrinks when it becomes the high temperature than 32℃. Therefore, the liposome that modified NIPAM becomes unstable and is broken at the time of high temperature than 32℃.</br> Reference</br> http://www.sigmaaldrich.com/etc/medialib/docs/SAJ/Brochure/1/j_recipedds2.Par.0001.File.tmp/j_recipedds2.pdf</br></br>

<h5>Method</h5> ・Cork both of them and put in a refrigerator in half day. </br> We put observation buffer and fluorescence reagent in their tubes, and perform operation of ① and ② respectively. </br> ① Setting a supersonic wave device to 20℃ and scratching the supersonic wave for 15 minutes. </br> ② Setting a supersonic wave device to 40℃ and scratching the supersonic wave for 15 minutes. </br> We took 5μL of each mixed things and dilute them in observation buffer of 195μL and collected appropriate amount of object and observing them. </br>

<img src="http://openwetware.org/images/f/fc/PNIPAM-phospholipid.png" width="400"></br>

    Fig4 PNIPAM付き脂質</br></br>

<h5>Result</h5> From Fig (?) and Fig (? + 1) when the temperature is 32℃ or low, liposome were observed. But when the temperature is higher than 32℃, liposome were not observed.</br> <table>

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  <img src="http://openwetware.org/images/7/72/Exp-2-00.png" width="400">
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  <img src="http://openwetware.org/images/d/d3/Exp-2-01.png" width="400">
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</table>

<h5>Discussion</h5> This time we observed liposomes respectively on the same condition excepting temperature.</br> So the next time we will try to observe liposomes that are made at lower temperature than 32℃. After observation, heat them at higher temperature than 32℃ and then, observe them whether there are any liposomes or not.</br>

<h4>3) Measurement of density of EGTA and time necessary to dissolve alginc acid gel</h4> <h5>Purpose</h5> It is necessary for the liposome in the alginic acid membrane to hold enough EGTA (one of the chelating agents) to dissolve alginic acid gel. In addition, when liposome is broken by an effect of NIPAM, Urea annealing and the destruction of the alginic acid film happen at the same time. But Time for Urea diluting Annealing must be shorter than Time for destruction of the alginic acid membrane. Therefore We measure density and time of the EGTA necessary to dissolve alginic acid gel beads.</br>

<h5>Method</h5> We make alginic acid gel beads and add 50mM,100mM,500mM EGTA to the solution. In addition, we prepare the thing which does not put EGTA in solution(control experiment)</br> We measure how many alginic acid gel beads decrease as time passes about the density of four kinds of EGTA.We take out the sample 0 minutes later, five minutes, ten minutes later・・・and count the number of alginic acid gel beads.We did the same experiment several times. </br>

<h5>Result</h5> From these results, the density of EGTA necessary to dissolve alginic acid gel beads is ?mM. and it took about ? minutes until alginic acid gel beads melted</br>

<h5>Discussion</h5>

<h4>4) Urea diluting annealing</h4> <h5>Purpose</h5> It is necessary for trigger DNA origami to be formed by Urea diluting Annealing. In addition, Time for Urea diluting Annealing must be shorter than Time for destruction of the alginic acid membrane to realize this system (system of the alginic acid group) . Therefore we measured the time that Urea diluting Annealing takes.</br>

<h5>Principle</h5> Polarity of H2O molecular becomes weak in the presence of urea. So urea interrupts the hydrogen bond of DNA base. For that, the melting point of DNA decreases. This enables hybridization at low temperature by decreasing the concentration of urea gradually. So, we can do annealing by diluting urea.</br>

<h5>Method</h5> We added M13mp18 and staples at the rate of 1:20 in TAE buffer with urea (6M) and Mg2+ (12.5mM).Then, we set the filter to floater and float it on TAE buffer with Mg2+(12.5mM).The devise was mixed by stirrer for 4 hours. By doing that, urea passes the filter and escape to outside buffer but DNA remains in filter, so we can do urea diluting annealing. Then, we observed sample remained in the filter by AFM and electrophoresis.</br> We observed structures as we designed by AFM imaging. </br>

<h5>Result</h5> The result is Fig5 as below. The scale of DNA origami is similar to our design. (for details of DNA origami design click here).</br>

    Fig5 尿素アニーリングにより作製したDNAオリガミのAFM画像（サンプル）</br></br>

<h5>Discussion</h5> We observed DNA origami like we designed as you can see Fig? .However, we also observed a lot of sheet structures like fragment. It is expectable that the reason why these fragments formed is that any staples didn’t hybridize with M13. We think this low yield of objective DNA origami was caused by high speed of diluting urea because in this experiment, urea easily passes filter. In our objective system, urea is diluted by destruction of liposome caused by the effect of PNIPAM. So, the speed of diluting urea is later than the speed of this experiment. It is necessary to measure the yield of DNA origami when the speed of diluting urea is later.</br>

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<h4>5温度を上げればアルギン酸膜が破壊されることの確認</h4></br>

2と3の実験が成功したのでこれらを組み合わせて、温度を上げて内部にキレート剤であるリポソームが割れれば、アルギン酸膜が割れるかどうかを調べるために以下のような実験を行った。  </br>

まず、アルギン酸ゲルビーズを作製して位相差顕微鏡で30μℓ当たりのアルギン酸ゲルビーズの数を数えた。</br> 次に、割れた後に系全体のＥＧＴＡの濃度が実験3で調べた最適な濃度になるような量のＥＧＴＡを入れたリポソームを作製した。これをアルギン酸ゲルビーズの入っている溶液の中に入れて温度を約３２度に上げた。その後、位相差顕微鏡で30μℓ当たりのアルギン酸ゲルビーズの数を数えた。（結果を表で示す）</br> ＥＧＴＡ付きリポソームをいれて温度を上げた後の方がアルギン酸膜の数が減っていたので、温度を上げることでニッパム付きのリポソームが破壊されて、キレート剤であるＥＧＴＡが放出されてアルギン酸膜が破壊されたと考えられる。</br></br>

<h4>6アルギン酸膜内で尿素アニーリングができていることの確認</h4></br>

1と4の実験が成功したのでこれらを組み合わせて、アルギン酸膜内で尿素アニーリングができるかどうかを調べるために以下のような実験を行った。</br> 二重ノズルの外管に1.5%アルギン酸ナトリウム溶液を、内管に尿素とＤＮＡオリガミの材料を入れてアルギン酸膜を作製した。時間をおいてキレート剤を加えて、アルギン酸膜を破壊した。溶液をとってＡＦＭで確認した。（ＡＦＭの画像）</br> 設計したとおりのＤＮＡオリガミが観察されたのでアルギン酸膜内で尿素アニーリングができたと考えられる。</br></br>

<h4>7全体のシステムの機能確認</h4></br>

5と6の実験が成功したのでこれらを組み合わせて、我々が目指しているシステム全体が機能しているかどうかを調べた。</br> まず、内部に尿素と、ＤＮＡオリガミの材料と、キレート剤であるＥＧＴＡを入れたニッパム付きリポソームを作製する。次に、二重ノズルを使ってアルギン酸膜を作製する。位相差顕微鏡でアルギン酸膜の数を数える。温度を約３２度に上げる。もう一度アルギン酸膜の数を位相差顕微鏡で数えた。また、同じ溶液をＡＦＭで観察した。</br> 温度を３２度に上げる前と後で、アルギン酸膜の数は減少してその溶液からＤＮＡオリガミがＡＦＭで観察できたので、私たちの目指しているシステムが機能していると考えられる。</br>

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     </article>

     <article data-title="Chain Reaction">

<h3>Step 2 Chain-reactive burst</h3> <p>Once the trigger DNA, which begins the interaction, is released, the next is the chain-reactive burst. If a liposome containing new triggers and active ingredients is broken, the released triggers come to destroy the surrounding liposomes one after another.<br> We tackled the problem of destroying liposomes by the following two approaches.</p> <br> <ur><li>ⅠBending membranes</li> <li>ⅡUtilizing flower micelles</li></ur> <br> <h4>ⅠBending membranes</h4> <h4>Experiment list</h4> The experiment necessary for realization of Bending membrane is following.<br> 1)Making DNA origami<br> 1-1)Making DNA origami<br> 1-2)Labeling DNA origami<br> 2)Destroying liposomes<br> 2-1)Making liposomes<br> 2-2)Investigating the interaction of DNA origami and liposomes<br> 2-3)Counting liposomes<br> <br> <br> <h4>1)Making DNA origami</h4> <h4>1-1)Making DNA origami<h4> <h5>Purpose</h5> In our project, we used DNA origami as triggers for destroying liposomes. We designed a rectangular DNA origami with a chipped edge and tried to make it.<br> <br> <h5>Principle</h5> DNA origami is a method applied to making nano-structures of various shapes. DNA origami consists of two kinds of strands: scaffold and staples. Scaffold is a long round single-stranded DNA, and staples are short linear single-stranded DNAs.<br> <br> <h5>Method</h5> We mixed M13mp18, staples, 5xTAE Mg2+, and mQ in a microtube and annealed it for 2.5 hours.<br> <A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br> <br> <h5>Result</h5> We confirmed that our DNA origami was well formed by AFM (Atomic Force Microscope) (Fig.1).<br> <Img Src="http://openwetware.org/images/d/d9/Outsideafm2.png"> <br> Fig.1 AFM image of DNA origami (M13: 4nM, staples:20nM)<br> <br> <h5>Discussion</h5> Just like our design, rectanglar origamis with chipped edges were observed.<br> <br> <br> <h4>1-2)Labeling DNA origami<h4> <h5>Purpose</h5> If the origami is fluorescently labeled, it is much easier to observe the effect of DNA origami on liposomes. So we labeled our origami by hybridizing it with fluorescent tagged DNA strands.<br> <br> <h5>Method</h5> Our DNA origami has many staples that can bind to fluorescent tagged DNAs for labeling. We mixed fluorescent tagged DNAs together with DNA origami staples in annealing solution.<br> In addition, to see if the origami binds to the fluorescent tagged DNA in shorter time, we added the fluorescent tagged DNA into control annealing solution, which contained no fluorescent tagged DNA, and left it for 40 minutes.<br> To see the origami was well labeled with fluorescent molecules, we used electrophoresis. <br> Electrophoresis was conducted with a 1% agarose gel, CV100V for 50 minutes.<br> <A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br> <br> By scanning a gel before staining, we can see only the bands of DNA structures with fluorescent molecules; scanning a gel after staining, we can see the bands of all DNA structures. So we scanned a gel before and after staining (we scanned both a non-stained and a stained gel). <br> First we saw the bands of our origami in a non-stained gel. Then, we compared the bands with those in a stained gel. If the bands of origami in a non-stained gel were at the same height as those in a stained gel, we can say that our origami is successfully fluorescently labeled.<br> <br> <h5>Result</h5> In a non-stained gel (Fig.2), only bands in lane 3 and 4 from the left (*Ori, **Ori) can be seen. They are fluorescent labeled structures. In addition, as they gave the same result, 40 minutes is long enough for fluorescent labeling.<br> <Img Src="http://openwetware.org/images/0/01/Outside-gel-3.png" width="300"><br> Fig.2 Non-stained gel image: only bands in two lanes can be seen. From the left, they are DNA origami with fluorescent molecules in pre-annealing (Ori*), and DNA origami with fluorescent molecules in post-annealing (Ori**)<br> <br> In a stained gel (Fig.3), maker (lane 1) had the longest DNA strand of 20kb. Comparing this and M13mp18 (lane 2) with annealed DNA origamis (lane 3,4,5), the bands of the origamis are at the higher position. Therefore, we concluded that in lane3~5, DNA origami structure made of M13 and staples were made as we had expected. <br> We considered that the bands in lane3~5 are seen as if they were diffused, just because our origami has many staples binding to the fluorescent tagged DNAs, and each origami attaches to different number of them, and its molecular weight varies.<br> <Img Src="http://openwetware.org/images/c/c6/Outside-gel-2.png" width="300"> </br> Fig.3 Stained gel image: from the left, maker, M13mp18, Ori*, Ori**, and DNA origami with no fluorescent molecule (Ori)<br> <br> <h5>Discussion</h5> Combining the results of Fig.2 and 3, the fluorescent labeled bands in lane3 and 4 in Fig.2 are at the same height as those of DNA origami in Fig.3. Thus, we concluded our origami was successfully fluorescently labeled.<br> <br> <h4>2)Destroying liposomes</h4> <h4>2-1) Making liposomes</h4> <h5>Purpose</h5> We make liposomes that are to be broken by DNA origami.<br> <br> <h5>Principle</h5> Phospholipids, which compose liposomes, are amphipathic molecules. They have hydrophilic and hydrophobic groups, and when they touch water, they make micelles: some hydrophilic groups take water inside. At the same time, other hydrophilic groups touch the water outside. So they form the innermost and outermost part of a micelle. On the other hand, the hydrophobic groups form the intermediate part of a micelle. <br> In this way, spherical liposomes are made.<br> <br> <h5>Method</h5> To make liposomes, first we mixed lipid (DOPC) and solvent (CHCl3) in a microtube, and desiccate it with Argon gas. Then, adding some buffer (1xTAE Mg2+), we heated it in warm water for a few hours.<br> <A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br> <br> The result and discussion are integrated in the next passage of (2-2) Investigating the interaction of DNA origami and liposomes.<br> <br> <br> <h4>2-2) Investigating the interaction of DNA origami and liposomes<h4> <h5>Purpose</h5> To destroy liposome with our origami, first we investigated how our DNA origami affected liposomes.<br> <br> <h5>Principle</h5> To destroy liposomes with our origami, many origamis have to hybridize with the surface of liposomes.<br> To begin with, we added cholesterol-conjugated single-stranded DNAs (in the rest of this document, referred to as ccDNAs) into liposomes, and made them float on the surface. If the ccDNA have a complementary part to our origami, the origami is expected to hybridize with the surface. In this way, many origamis would hybridize with liposome via ccDNAs.<br> <br> <h5>Method</h5> We added ccDNAs into liposomes at the final concentration of 0.018, 0.069, 1.8, and 6.9µM. Then we observed the samples with a phase microscope. Next, adding fluorescently labeled DNA origamis into the above liposomes, we saw if some change would happen with a fluorescent microscope.<br> <A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br> <br> <h5>Result</h5> In all four conditions, liposomes were observed with a phase microscope. We confirmed the formation of multilamella liposomes (Fig.4~7).<br> <br> <Img Src="http://openwetware.org/images/7/72/Lipofig4.png" width="400"></br> Fig.4 Phase microscope image of liposomes (cholesterol-conjugated DNA: 0.018µM)<br> <br> <Img Src="http://openwetware.org/images/d/d0/Lipofig5.png" width="400"></br>

Fig.5 Phase microscope image of liposomes (cholesterol-conjugated DNA: 0.069µM)<br>

Fig.6 Phase microscope image of liposomes (cholesterol-conjugated DNA: 1.8µM)<br>

Fig.7 Phase microscope image of liposomes (cholesterol-conjugated DNA: 6.9µM)<br>

<br> Adding fluorescently labeled DNA origamis into the above liposomes, we saw if some change would happen with a fluorescent microscope.<br> When the concentration of ccDNA was 0.018, 0.069µM, many gleaming (in green color) liposomes were observed. We confirmed that the fluorescently labeled origamis well hybridized with the liposomal surface (Fig.8,9,10). <br> <table>

<tr>
 <td>
  <Img Src="http://openwetware.org/images/6/6c/Lipofig8.png" width="400">
 </td>
 <td>
  <Img Src="http://openwetware.org/images/a/a6/Lipofig9.png" width="400">
 </td>
</tr>

</table> Fig.8,9 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 0.018µM)<br> <Img Src="http://openwetware.org/images/b/b4/Lipofig10.png" width="400"></br> Fig.10 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 0.069µM)<br> <br> On the other hand, when the concentration of ccDNA was 1.8µM, few gleaming liposomes could be seen with a fluorescent microscope (Fig.11). This result indicates the possibility that liposomes were broken.<br> <Img Src="http://openwetware.org/images/1/18/Lipofig11.png" width="400"></br> Fig.11 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 1.8µM)<br> <br> When the concentration of ccDNA is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.12).<br>

Fig.12 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 6.9µM)<br>

<br> <h5>Discussion</h5> From these results, we put forward the following hypothesis about the interaction of DNA origami and liposomes.<br> When the concentration of ccDNA is low (0.018, 0.069µM), DNA origamis hybridize with the surface of the liposomes relatively stablely. When the concentration is middle (1.8µM), more DNA origamis hybridizes with the surface and place stress on it. Then, liposomes become fragile and easy to be broken. When the concentration is high (6.9µM), some liposomes exist individually, and others form networks via ccDNA and DNA origami complexes.<br> <Img Src="http://openwetware.org/images/7/7c/Experimentinsidefig.png"><br> <br> According to this hypothesis, when the concentration of ccDNA is 1.8µM, DNA origami destroys liposomes. Therefore, in the following experiment, we checked if DNA origami would destroy liposomes at this concentration of ccDNA.<br> <br> <br> <h4>2-3)Counting liposomes</h4> <h5>Purpose</h5> To see if DNA origami destroys liposomes, we counted the number of liposomes before and after adding DNA origami. <br> <br> <h5>Method</h5> For the sake of observation convenience, we mixed TR-DHPE (red fluorescent dye) with lipid (DOPC) and solvate (CHCl3), and made liposomes. The liposomal surfaces were dyed by TR-DHPE.<br> Then we added ccDNAs at the final concentration of 1.8µM, and counted the number of liposomes with a fluorescent microscope.<br> After counting, we put DNA origami and counted the number of liposomes again.<br> <A href="http://openwetware.org/wiki/Biomod/2013/Sendai/protocol">Protocol</A><br> <Img Src="http://openwetware.org/images/a/a3/Addingimage.png"></br> <br> <br> <br> <h4>ⅡUtilizing flower micelles</h4> <h4>Experiment list</h4> The experiment necessary for realization of Bending membrane is following.<br> 1) Making liposome <br> 2) Confirming the formation of loop structure by SPR<br> 3) Confirming the hybridization of trigger and loop DNA.<br> 4) Destroying liposome<br> <br> <br> <h4>1)Making liposome</h4> <h5>Purpose</h5> We make liposomes that are to be destroyed by flower micelle method.<br> <br> <h5>Principal</h5> We made normal liposomes made of DOPC and phase-separatied liposomes made of DOPC, DPPC and cholesterol.<br> Phase-separated liposomes are liposomes made by several kinds of lipids. On the surface of phase-separated liposomes several kinds of lipids separate and the liposomes are formed by some layers.<br> As the surface lipids of the phase-separated liposomes are not so changeable as the normal liposomes, It is considered that power produced by the hybridization of the loop and trigger strands reaches the liposome more effectively.<br> So the phase separation liposome was used for experiments this time.<br> <br> <h5>Method</h5> <ur><li>1. Making DOPC, DPPC, and Cholesterol lipid Lipid<br> 1-1 Put 7.8 mg DOPC, 7.3mg DPPC , and 3.8mg Cholesterol into each microtube, and add 1ml CHCl3.<br> 1-2 Put it in a ultrasonic bath of 60 degrees Celsius for one hour.<br> 1-3 10mM DOPC, DPPC, Cholesterol lipid is made.<br></li>

          <br>
           <li>2. Making phase-separated liposomes<br>
           2-1 Mix DOPC,DPPC, and Cholesterol at the ratio of 1:1:1 to make phase-separated liposomes. In this experiment, mix 4µl DOPC (10mM), 4µl DPPC (10mM),4µl Cholesterol (10mM) and 88l buffer well.<br>
           2-2 Add 12µl Texas red (10M) <br>
           2-3 Dry the sample using Argon gas<br>
           2-4 Hydrated the dried sample with by 100ml 1xTAE<br>
           2-5 Put the sample in hot water for three hours. Then leave it at low temperature for one hour to let the surface lipid separate.</il></ur><br>

<br> <h5>Result</h5> As is shown in Fig.13, phase-separated liposomes were observed by a fluorescent microscope. They are basically multi-lamella liposomes.<br> We confirmed the formation of phase-separated liposomes with a fluorescent microscope.<br>

<Img Src="http://openwetware.org/images/7/79/Flowerex1.png"></br> Fig.13 Fluorescent microscope image of phase-separated liposomes<br> <br> <h5>Discussion</h5> Using the above-mentioned method, we successfully made phase-separated liposomes.

<h4>2) Confirming the formation of loop structure by SPR</h4> <h5>Purpose</h5> To destroy liposomes by flower micelle method, we aim to attach many loop strands to the surface of liposomes. <br> To achieve this, we adopt the same hybridization method via ccDNAs as used in Ⅰbending membranes into liposomes: the ccDNA has a complementary part to our loop strand and the loop strand is expected to hybridize with liposomes.<br> We checked the hybridization of liposomes and ccDNAs, and that of ccDNAs and our loop strands. <br> <br> <h5>Principle</h5> As our loop strand is too small to observe with an AFM or a fluorescent microscope, we used an apparatus called SPR.<br> SPR is a Surface Plasmon Resonance assay that estimates the weight of molecules attached to membrane surface, by the change of the reflection of the laser beam.<br> If ccDNA attaches to a liposome, and then loop strand attaches to it, SPR value increases after each step.<br> We measured SPR value after each step of adding DOPC into liposomes, and loop DNAs into it.<br> <br> <h5>Method</h5> <ur><li>1. Inject 45µl DOPC (100mM) into SPR</li> <li>2. Inject 5µl NAOH to SPR in order to stabilize the point</li> <li>3. Inject 10µl ccDNA (0.1M) to SPR</li> <li>4. Inject 10µl loop DNA of 40 bp (0.1M) to SPR</li> <li>5. Inject 10 µl trigger DNA of 40 bp (0.1M) to SPR</li> <br> <br> <h5>Result</h5> The result was shown in Fig.14 below.<br>

<Img Src="http://openwetware.org/images/f/fd/Flowerex2.png"></br> Fig.14 The transition of SPR value<br> <br> As the first injection of ccDNAs caused no change of SPR value, we injected ccDNAs for two times. <br> Fig 14 shows that SPR value increased after injecting ccDNAs and loop DNAs. Moreover, we should note that after injecting trigger DNA, some changes of SPR value were observed.<br> <br> <h5>Discussion</h5> Fig.14 shows the behavior of materials on the surface of liposomes. The increase of SPR value after injecting ccDNAs indicates that ccDNAs successfully combined with liposomes. Similarly, it is considered that loop DNAs combined with ccDNAs. <br> Thus, we confirmed the formation of the loop structures on liposomes.<br> <br> <br> <h4>3) Confirming the hybridization of trigger and loop DNA</h4> <h5>Purpose</h5>

We checked whether trigger DNA hybridizes with loop DNA at normal temperature by electrophoresis. <br>

<br> <h5>Method</h5> <ur><li>1. Prepare three microtubes and put three kinds of trigger DNAs (10, 20, 40b; 5µl, 100nM) into each tube.</li> <li>2. Add three kinds of loop DNAs (10, 20, 40b; 5µl, 100nM) into corresponding tube (tube that contains trigger DNA of corresponding number of nucleotides) and leave them at normal temperature for approximately one hour.</li> <li>3. Add 6x loading buffer with the quantity of 20% of the samples.</li> <li>4. Make an acrylic amide gel.</li> <li>5. Load samples (including maker) into 10 lanes</li> The electrophoresis was conducted with CV 100V for one hour.<br> <br> <h5>Result</h5> The result was shown in Fig.15.<br>

<Img Src="http://openwetware.org/images/3/37/Flowerex3.png"></br> Fig.15 Stained gel image<br> <br> The lane of 20 base loop and trigger shows a strong band at different height from the band of only 20 base loop and trigger. As for the samples of 40 base, the result was the same. <br> On the other hand, the lane of 10 base loop and trigger shows a band at the same height as the band of only 10 base loop. No band was seen in the lane of only 10 base trigger.<br> <br> <h5>Discussion</h5> The fact that the band of 20 base loop and trigger was at the different height from the band of only 20 base loop or trigger indicates that 20 base loop and trigger DNA hybridized and made a double strand. As the samples of 40 base showed the same result, we concluded that 20 and 40base loops and triggers hybridize at normal temperature.<br> However, as for the samples of 10 bases, there was no difference between the two band height. Therefore, 10 base loop and trigger had not hybridized. <br> It is estimated that no band was seen in the lane of only 10 base trigger because of some kind of mistakes. Therefore we do not take this into consideration.<br> From the above, we find that the 20 and 40nt trigger hybridizes with a loop at normal temperature.<br> <br> <br> <h4>4) Destroying liposome</h4> <h5>Purpose</h5> It was tested if liposomes would be destroyed by adding trigger DNA.<br> <br> <h5>Principle</h5> Whether liposomes are destroyed or not can be decided by counting the number of liposomes before and after the trigger addition. As a control, we added the same amount of buffer instead of trigger. Liposomes are observed by a fluorescent microscope.<br> <br> <h5>Method</h5> <ur><li>1. Make liposomes with loop DNAs<br> 1-1 Mix 2µl liposome (0.2mM) with 2µl ccDNA (10µM) at normal temperature<br> 1-2 Add 2µl loop DNA (20µM)</li><br> <li>2. Destroy the liposomes with the loop DNAs<br> 2-1 Add 2µl trigger DNA (20µM) </li></ur><br> <br> <h5>Result</h5> Fig.16 is the result of the control experiment; Fig17, the result of adding trigger DNAs.<br>

  <Img Src="http://openwetware.org/images/4/4f/Flowerex4.png">
  <Img Src="http://openwetware.org/images/1/1e/Flowerex5.png">

Fig.16,17 Fluorescent microscope image of liposomes <br>(Fig.16: control, Fig.17: sample added trigger DNAs)<br>

As it was difficult to count the number of liposomes in both cases, we did not count them.<br> <br> <h5>Discussion</h5> As we were not able to see a clear numerical change, we did not see whether liposomes were destroyed by this experiment.<br> Two ideas why liposomes were not destroyed are come up:<br> 1. The lipid ratio for making liposomes was not appropriate. We should investigate the most appropriate and effective ratio for destroying liposomes.<br> 2. Liposomes in this experiment were multi-lamella ones: Multi-lamella liposomes have some leaflets piled up. It is considered that more power is needed to destroy them. We would try other methods except the hydration method in future to make uni-lamella liposomes (which is relatively easy to destroy).<br> Solving the above- mentioned problems, liposomes would be destroyed<br>.

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