Step1 Egg-type initiator
Experiment listThe experiment necessary for realization of Egg-type initiator is following 1-1) Making liposome in alginate hydro gel beads 1-2 ) Preparing of alginate hydro gel membrane containig buffer 2) Function confirmation of PNIPAM 3) Measurement of density of EGTA and time necessary to dissolve alginc acid gel 4) Urea diluting annealing
1-1) Making liposome in alginate hydrogel beads
PurposeWe need to put liposome in alginate hydrogel beads for chelating agent which in the liposome collapse alginate hydrogel membrane after liposome is collapsed. at first We make liposome and put it in a sodium alginate water solution and make alginate hydrogel beads with the solution
MethodWe 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 Fig.1 for 2-3 minutes Fig.1 Experimental device of alginic acid gel beads
ResultBecause 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.
DiscussionFrom Fig.2, 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.
1-2 ) preparing of alginate hydro gel membrane containing buffer
PurposeViscosity 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.
PrincipleWhen 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.
MethodWe used double capillary(Fig.2), 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.
Fig.3 Structure of double capillary
ResultWe observed alginate hydrogel by confocal laser microscope and we could confirm fluorescence in the alginate hydrogel.
|Fig.4 Phase contrast microscope image of alginate hydrogel membrane||Fig.5 Fluorescent microscope image of alginate hydrogel membrane|
DiscussionAlginate 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.
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.
2) Function confirmation of PNIPAM
PurposeIn this project, it is necessary that liposome collapses surely when we increase temperature to 32℃.So, we confirmed whether liposome with PNIPAM collapses when the temperature become 32℃.
PrincipleNIPAM 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 collapsed at the time of high temperature than 32℃.
Fig.6 Function of PNIPAM
Method・Cork both of them and put in a refrigerator in half day.
We put observation buffer in their tubes, and perform operation of ① and ② respectively.
① Setting a supersonic wave device to 20℃ and scratching the supersonic wave for 15 minutes.
② Setting a supersonic wave device to 40℃ and scratching the supersonic wave for 15 minutes.
We took 5μL of each mixed things and dilute them in observation buffer of 195μL and collected appropriate amount of object and observing them.
Fig.7 Phospholipid decorated with PNIPAM
ResultFrom Fig.8 and Fig. 9 when the temperature is 32℃ or low, liposome were observed. But when the temperature is higher than 32℃, liposome were not observed.
|Fig.8 Phase contrast microscope image of liposome(20℃)||Fig.9 Phase contrast microscope image of liposome(40℃)|
DiscussionThis time we observed liposomes respectively on the same condition excepting temperature.
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.
3) Measurement of density of EGTA and time necessary to dissolve alginc acid gel
PurposeIt 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 collapsed 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.
MethodWe 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)
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.
ResultFrom 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
4) Urea diluting annealing
PurposeIt 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.
PrinciplePolarity 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.
MethodWe 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.
We observed structures as we designed by AFM imaging.
Fig.10 Method of urea diluting annealing