The Team Sendai B
The Team Sendai had two teams, Team Sendai A and B, until Biomod Japan Meeting 2012. After that, the two teams were integrated based on Team Sendai B. This is a diary of the Team Sendai B.
Since the stage of reading and studying various papers finished, our activity as a Kisozemi team (basic seminar course for first year undergraduate students) completely started from today. We scheduled our future activity, then we discussed what kind of molecular robot to make.
We brainstormed about our robot and learned how to use the micro-pipette. We will discuss our robot more realistically on the next week.
Each member chose 1 or 2 interesting ideas from the last week’s brainstorming. Then we expressed and discussed about them. At the end, we decided that our project is composed of 2 systems. One was a "doorman" system that opens the lid of a DNA origami box when the system catches the target. The other was a "porter" system that carries a target into a DNA origami box. We divided our team into 2 groups and agreed on presenting about each topic next time after a careful consideration.
We had a presentation about each team’s thoughts, doorman team and porter team. According to the result of presentation, we conclude that we need to think about a better structure. We talked about which structure is the best. We will have a presentation on today’s subject next week.
Today, we did our 2nd presentation. Through this presentation, we roughly decided what robot to make. We will make a robot which carries a target into itself. Next week, we will think about the best structure of our robot and we will do the 3rd presentation.
We performed the 3rd presentation. The details appear in this page.
In this presentation appeared the issue of deciding the size of the target and the robot. We are going to do the 4th presentation including the robot’s size next Monday.
We did the 4th presentation. We learned that we must explain why we want to capture a target. Presentations require the complete story for convincing other persons. Moreover, since the design of a robot was becoming clear, we begun the full-scale design. We thought that the shape of our robot should be a pipe. Therefore, we learned how to use caDNAno. Each member task is to design the robot's tube that will capture the target by next week.
We had the fifth presentation. We split into three groups, Wiki team, robot design team, slide & script team. Robot design team started to try designing a structure of the size precisely determined on today’s meeting.
Today, we earnestly started to design the robot's tube. We can design our pipe based on a previous DNA origami pipe by Dr. Shawn Douglas. So we will create a new design by next week. Next time, we will learn how to do the electrophoresis.
We had a meeting, designed the robot structure with caDNAno and we did the electrophoresis as an exercise using some laboratory samples.
Today, we designed our loop and tournament structures with caDNAno. Loop and tournament structures are Biomod2012 Basic Seminar’s original structure. The details appear in Wiki and make a schedule for experiments. We understood that making and observing a tube is difficult, so we give up our design up to now and decide to make new things. We calculated the melting temperature (Tm) of the loop structure and tournament structure, and determine arrangements. It was ordered and will come until the day after tomorrow.
Today, we carried on the electrophoresis of M13 and a field we made last year as an exercise for the experience on loop and tournament structures . As a result, we probably succeeded the annealing process because a staple with M13 moved slowly and was appeared in the correct band position between 7000~5000 from the ladder. This concentration was too dark because it was in 0.8 μℓ 6X Loading buffer and 4.2 μℓ sample. We should do them with 1 μℓ of loading buffer next time. We redesign a triangular tube structure and would complete it by 8/9. We would receive samples which have the loop and the tournament structures and start a demonstration of them tomorrow.
Today, we designed a tube structure. We asked for evaluation of the design of the tube with the triangular prism to teacher Hamada, and he said that it was not completed yet. Then, we are going to keep designing the tube with the hexagonal prism and the tube with the triangular prism in parallel. Also, we prepared the experimental of loop and tournaments structures. We decided to start the experiment. First of all in preparation, we created a 20% acrylamide gel, then stored it on the refrigerator. In addition, we received the sample and anneal it.Tomorrow we would do the experiment of electrophoresis to prove our hypothesis.
We did the electrophoresis with 10% and 20% acrylamide gel. The details of experiment is in the experimental result page. We can’t find the bands in the 10% gel. Maybe because of using Midori Green as a stain. On the other hand, We use SYBR Gold as a stain for 20% gel. We could look the bands clearly, but the result was not what we want. On 8/17, we will do our experiment with changing the conditions.
O-bon vacation ――August 10th-16th――
Today we agreed some points to improve the experiment which did not go well with the experiment on 8/9. We change to sybr gold staining of electrophoresis on 10% acrylamide gel and extend to 50 minutes, the time interval for aggregating of Query and tournament or loop n tube. We failed to make the gel and we took a lot of time to do the experiment. Since we recreate the solution , we could not have sufficient time for experiments. Thus, we did only experiment of 10% gel today. We keep the 20% gel, but I thought better to rebuild them because the bubbles of steam appeared during production. I wonder whether appearance of bubbles with steam might be normal. The photo was published in the Google drive of the results of electrophoresis. The band did not appear clearly. This is likely because of a failure on making the gel. Possible causes for the failure are, using the old acrylamide, forgetting to put a buffer which dilute the sample.
We tried again yesterday's electrophoresis experiment by changing conditions.
1. We did the electrophoresis of the gel before we put our samples.
2. We did the electrophoresis on the refrigerator.
3. We did the electrophoresis at 50V.
1 will lead our experiment to succeed. 2 prevent Query,Loop, Tournament1 and Tournament2 fall to pieces. We were careful to not forgot to put buffer so as not to fail our experiment. At first, we did at 50V, but it took long time, so we changed to 100V for 1.5 hours.
We did the electrophoresis 10% gel for Tournament1 hours and 20% gel for Tournament2 hours. In 10% gel, the bands appeared clearly. Sample in which Query, Loop and tournament1 mixed showed that Query combined with Tournament2, and Sample in which Query, Loop , tournament1 and tournament2 mixed showed that Query combined with Tournament2. Considering those, Query was likely to move from Loop to Tournament1, or to move from Tournament2 to Tournament3. However, in Sample in which Query, Loop and Tournament1 mixed, the band of Query and Loop , and the band of Loop and Tournament1 appeared very clearly both. So we suspected that Query actually moved from Loop to 3.
In Sample in whichQuery, Loop, Tournament1 and Tournament2 were mixed , we couldn’t find the band around Loop. So we may have forgotten to put Loop. Because of it, we weren’t sure moving from Loop to Tournament1 . But, we were sure from Tournament1 to Tournament2. In 20% gel, we couldn’t find the band clearly. Because of it, we will use only 10% gel next time.
First, we made two 10% gels. We saved one in the refrigerator and we did the electrophoresis with the same condition of yesterday. We did the electrophoresis at 50V for Tournament1 hours. Considering from the result, Query certainly moved from Tournament1 to Tournament2. In Sample in which Query,Loop and Tournament1 were mixed, the band of Loop appeared weakly, but the band of Query and Tournament1 appeared lower, so Query seemed not to combined with . The factor of it seemed to be the interaction between Loop and Tournament1.
We did the electrophoresis with changing sample’s combination. Today’s sample was the previous combination Query, Loop, Tournament1, Tournament2, mixing Query and Loop, mixing Query and Tournament1, and the new combination mixing Loop and Tournament1, mixing Loop and Tournament2, mixing Tournament1 and Tournament2. We used 10% gel, and 1×TAE was added to Mg2+ as a buffer. Considering from the result, Query seemed to combine with Loop, and Query seemed to combine. But, Loop seemed to combine with Loop, Tournament1 seemed to combine with Tournament1.
Today we made two acrylamide gels for tomorrow electrophoresis. We changed the design of the robot a little bit. Its each end has Loop and carry a target to middle of robot. We noticed that Loop and Tournament1 are likely to combine with each other or with themselves. Because of this, we changed all extra sequences of Loop and tournament into adenine. We ordered our new robot today and will arrive next Monday but, annealing will take long time, so we will use them after BIOMOD meeting in Japan. We uploaded some results of our experiments to Wiki. Tomorrow, We will upload Japanese version of our all data.
We did electrophoresis with the previous samples. This time we did it at room temperature. The result seems to confirm that Loop and Tournament1 combines not with Query but with themselves. We'll receive the improved design DNA of a Loop and Tournaments next Monday, and then do experiments when we have time. As we've made the explanation of experiments and diary in English to some extent, we'll start to translate the whole Wiki into English. We're making slides for the presentation too.
We received new " Loop " and "Tournament" DNA sample and dispensed them. We'll do experiment tomorrow. In addition, we made our wiki and our presentation slide.
We did experiment with new DNA. Mixing Query and Loop showed that query did not combined with Loop. We thought high temperature caused the result. We'll do electrophoresis in refrigerator tomorrow. In addition, we made our wiki and our presentation slide.
We did electrophoresis in the refrigerator today. Mixing Query, Loop , and Tournament1 showed that Query combined with Tournament1, and mixing Query, Loop, Tournament1, and Tournament2 showed that Query combined with Tournament2 but mixing Query and Loop was not showed that query combined with Loop. We'll do electrophoresis in the refrigerator with cool buffer tomorrow. In addition, we made our wiki and our presentation slide.
We did electrophoresis in a refrigerator (at Tournament2 degrees) with cool buffer today. As a result, we successfully see the Loop and the object combine. And Loop and tournaments don't interact each other. We practiced for BIOMOD meeting in Japan. Based on what we were said in today’s practice, We edited the outline of slides. We were also said that our structure’s name “ Loop ” and “tournament” was difficult for an audience to understand. So we changed both names into new name “Selector”. We'll do experiment to make sure of our success and continue to make the slides.
As yesterday, we did the electrophoresis in the refrigerator with cool buffer. But, because of the trouble with the gel, we failed our experiment. Also, we made our wiki and our presentation slide.
We corrected the manuscript of presentation and Wiki in English. We have already written these in English, so We should only rewrote these in English.
BIOMOD JAPAN MEETING 2012 on September 9th
Jpapan meeting was held at Tokyo university. The result is that Team Sendai B was 4th and Team Sendai A was 6th.
The All Team Sendai
Team Sendai A has been integrated into the Team Sendai B because B is superior to A in the ranking of the Biomod japan meeting. We work on same project together.
We did electrophoresis to confirm best time Target moves to Selecter3 from Selecter2. Mixed Target with Selecter2,and annealed. We left them different time 60,50,40,30,20,10,0 minutes after mixing Target+Selecter2 with Selecter3. But we did not get good result. Maybe we failed annealing, Target with Selecter2.
We did electrophoresis to confirm best time Target moves to Selecter3 from Selecter2. Mixed Target with Selecter2,and annealed. We left them different time 60,50,40,30,20,10,0 minutes after mixing Target+Selecter2 with Selecter3. We got good result. Best time was 10 minutes.
We made 10% acrylamide gel today. We annealed Target to Selector1 in Thermal cycler at one degree for 30 minutes. Then we added Selector2 when 0,10,20,30,40,50,60 minutes passed. We waited for them to react at the room temperature. We did electrophoresis in a fridge. Result: Target and Selector1 attached to each other (The annealing succeeded). But Target didn't move from Selector1 to Selector2. Conclusion: Target and Selector1 are likely to attach to each other too strongly. So tomorrow We won't anneal Target to Selector1. Instead, We'll just put Target and Selector1 together for one hour.
We made 10% acrylamide gel today. We mixed Target to Selector1 for one hour at the room temperature. Then we added Selector2 when 0,10,20,30,40,50,60 minutes passed. We waited for them to react at the room temperature. We did electrophoresis at the room temperature. Result: Target and Selector1 attached to each other. But Target didn't move from Selector1 to Selector2. We just don't know why Target didn't move from Selector1 to Selector2.
We made 10% acrylamide gel today. 1. We did the experiment with the same condition as yesterday's. (We mixed Target to Selector1 for one hour at the room temperature. Then we added Selector2 when 0,10,20,30,40,50,60 minutes passed. We waited for them to react at the room temperature. We did electrophoresis at the room temperature.) Result: Target and Selector1 attached to each other. But Target didn't move from Selector1 to Selector2. 2. We intended to see the body structure of hexagonal tube with AFM. But we decided not to do today because the SUM value was too low. 3. We did electrophoresis with a little different condition, to see whether Target moves from Selector1 to Selector2, and from Selector1 to Selector3. Result: Target moved from Selector2 to Selector3. But Target didn't seem to attach to Selector1.
We started the annealing of the gate that doesn’t have selector.
The annealing of the gate we started at September 22 had finished so we got 50μℓ in all.Then, we observed the sample of the gate by AFM using 5μℓ of the sample. Next, we purified 30μℓ of the sample on the way based on the monograph written by Shone.We show the process of purifying in the next; In this process, we do two section of purifying;the one using filter and the other using gels.
We continued the purifying.We cut the band of M13 and the gate with black light (for the reason to enable to see the place colored by SYBR Gold).Next,we moved each of them to tube and stroked the gel with the edge of scoopula.Then, we did centrifugation at 3000×g15°c for 3 min.Next, we moved each of the gels to DNA gel extraction spin column and did centrifugation at 3000×g15°c for 1 min.
We created 1×Folding Buffer and four 1%-agarose gels. We did electrophoresis with the gate purified by centrifugation.The result was there was a band at the place a little higher than M13.We suspect the band might be the gate.Furthermore,the band of M13 purified on the way based on the monograph written by Shone can been seen clearer than the band of M13 not purified. This shows the effect of purifying.
The staple we ordered for the reason to see the shape of the gate clearly by AFM arrived so we conducted dispensation.
We ordered new Selector1, new Selector3, new Selector4, and the new target with fluorescent molecule.
We purified the gate which have finished the annealing yesterday in the way based on the monograph written by Shone.
We did electrophoresis with the target, Selector, and mismatch.
We created a 0.7% agarose gel. We did electrophoresis with the gate purified in October 2.The band of gate with no Selector can be seen at the place little higher than the band of M13 but the band of gate with Selector cannot be seen at anyplace. We conducted dispensation of hydrophobic group.
We received the new target with fluorescent molecule. We did electrophoresis with the target, Selector, and mismatch.
We mixed the staple of the gate we will do annealing tomorrow.
We did electrophoresis with the target, Selector, and toehold.
We started annealing of the gate with hydrophobic group. The condition of annealing is 2min/-1℃ at 80℃～61℃ and 150min/-1℃ at 60℃～24℃.The concentration of Mg is 12.5mM.
We reconsidered the condition of annealing. We decided to change the concentration of Mg to 8mM and the speed of cooling (80°c~70°c and 40°c~25°c→2min/-1°c,70°c~40°c →repeat the next cooling :7.5min/+5°c then 7.5min/-6°c) and to try to do annealing based on the condition.
We did electrophoresis with the target with fluorescent molecule (we express it as *Target from now on), Selector, and mismatch.
We did annealing at the condition we decided yesterday.We annealed two types of gate;the one has new selector but doesn’t have neither hinge nor hydrophobic group and the other has both hinge and hydrophobic group but doesn’t have selector.
We did electrophoresis with *Target, Selector, mismatch, and toehold.
We did electrophoresis with *Target, Selector, and toehold.
We did electrophoresis with 5 samples of the gate which has hinge and hydrophobic group,new selector,3’ hydrophobic group,5’ hydrophobic group, and mixed 3’ and 5’hydrophobic group.The band annealed by new condition was pale so we cannot get good result.Furthermore, there might be mistake because the devise for annealing developed fever too much.Tomorrow,we are going to purify the gel we did electrophoresis today. We observed the sample of the gate with hinge and hydrophobic group(not purified).Then, we discovered slender structure and something crushed.
We did electrophoresis with *Target, Selector, and toehold.
We observed the samples by AFM again but we couldn’t discover the structure we found yesterday. The annealing conducted on the condition concentration of Mg is 12.5mM was started. We cut the bands from the gel we used for electrophoresis yesterday and purified them.
We did electrophoresis with *Target, the gate without selector, and the gate with selector inside.
The staples for connecting the gates arrived, so we dispensed them. We did electrophoresis to reconsider the condition of annealing but the result was not so good because of Mg contained in gel.We are going to do electrophoresis again by using the gel with no Mg.
We started annealing of the gate with new selector 1,the gate which can connect with other gates ,and the gate with 3’5’ hydrophobic group. We created 1% agarose gel which has no Mg and did electrophoresis the sample of the gate with connect-able gate,the gate with hinge and 3’ hydrophobic group, and the gate with 3 ’ hydrophobic group.The band of the gate with 3’ hydrophobic group was so clear so we conducted the gel purifying. By using the AFM, we observed the sample of the gate with 3’hydrophobic group we didn’t use for electrophoresis.
We filmed the you tube video for the first time. We started annealing of the connect-able gate.
We held luncheon meeting. We did electrophoresis by using the 1% agarose gel with no Mg.
We started the annealing on low concentration.The concentration of samples is 1nM and 10nM. We did electrophoresis of the sample that we started annealing on October 14.
We started annealing of the gates with selector1 only,selector 2 only,and both selector1 and selector 2. We did electrophoresis of the connect-able gate we started annealing yesterday.
We held luncheon meeting. We did electrophoresis to identify whether C1 and C2 behave correctly or not by using a 1% acrylamide gel.
We started the annealing of the connect-able gate. We did electrophoresis of C1 and C2 whose concentration is high.
We did electrophoresis for a material of our presentation.
We edited Experiment of Wiki.
We held luncheon meeting and talked about Wiki and presentation.
We did electrophoresis.
We practiced of presentation.
We did electrophoresis.