iGEM 2015 — Team SCUT-China

# Team:SCUT-China/Notebook

![](https://static.igem.org/mediawiki/2015/6/6d/2015-SCUT-China-loading2.svg)

# Loading

* ![](https://static.igem.org/mediawiki/2015/b/b9/2015-SCUT-China-logo.png)
* [HOME](https://2015.igem.org/Team%3ASCUT-China)
* [TEAM](https://2015.igem.org/Team%3ASCUT-China/Team)
  [+ Team Members](https://2015.igem.org/Team%3ASCUT-China/Team)
  [+ Attribution](https://2015.igem.org/Team%3ASCUT-China/Attributions)
* [PROJECT](Description)
  [+ Description](https://2015.igem.org/Team%3ASCUT-China/Description) [+ Experiments & Results](https://2015.igem.org/Team%3ASCUT-China/Experiments)
  [+ Protocols](https://2015.igem.org/Team%3ASCUT-China/Protocols)
  [+ Notebook](https://2015.igem.org/Team%3ASCUT-China/Notebook)
* [PARTS](https://2015.igem.org/Team%3ASCUT-China/Parts)
* [COLLABORATIONS](https://2015.igem.org/Team%3ASCUT-China/Collaborations)
* [HUMAN PRACTICES](https://2015.igem.org/Team%3ASCUT-China/Practices)
  [+ Overview](https://2015.igem.org/Team%3ASCUT-China/Practices) [+ Hospital Interview](https://2015.igem.org/Team%3ASCUT-China/hospitalInterview)
  [+ Seminar & Conference](https://2015.igem.org/Team%3ASCUT-China/Conference)
  [+ Data Analysis](https://2015.igem.org/Team%3ASCUT-China/dataAnalysis)
  [+ Law & Regulation](https://2015.igem.org/Team%3ASCUT-China/criticalThinking)
* [SAFETY](https://2015.igem.org/Team%3ASCUT-China/Safety)
* [MODELING](https://2015.igem.org/Team%3ASCUT-China/Modeling)

![](https://static.igem.org/mediawiki/2015/6/6d/SCUT-China-notebook-title.png)

### Friday 10/7

After finishing our final exam, we talked a lot about our project. We determined our goals and direction of getting there. In the discussion, work assignment had been done. Our team tidied the lab together. Moreover, with the help of our senior fellow-student brothers and sisters, we received lab safety training.

### Saturday 11/7

With the help of our senior fellow-student brothers and sisters, we studied the method of culturing cell. And we tried to culture some. Then we waited for the result.

### Monday 13/7

We were informed that we failed in culturing cells. We tried to recall the experimental process to find out the reason of failure. We found that maybe the overdue cell-culture medium we used caused the failure. We still wanted to try again, although not without pain.

![](https://static.igem.org/mediawiki/2015/d/d4/2015-SCUT-China-notebook-7-13.jpeg)

### Wednesday 15/7

We put forward experimental procedure and gave in detail.

### Sunday 19/7

Part of our team members took part in a meetup which was set up by Sun Yat-sen University Guangzhou, China. The meetup contained three parts: ice breaker, project presentation, world café. Many teams had been involved such as SKLBC-China, SKLBC-GDSYZX, SYSU-China, SYSU-Software, SCUT-China. In the meetup, they shared their ideas to us and we also introduced our project and showed our presentation to them. Their teacher and members pointed out our shortcoming and gave us some advices. And we also exchanged our views with modeling team to seek a better way to cooperation and development. We reaped a lot benefit from this meetup!

![](https://static.igem.org/mediawiki/2015/d/d4/2015-SCUT-China-notebook-7-19.jpeg)

### Thursday 23/7

We learned basics, as running a PCR, and we got every reagent we needed in our project. And we set those reagents on appropriate place. We can find everything we needed in the lab.

We set about designing three primers and came across some trouble. PDE5A mRNA has three variants, we felt confused about choosing which variant.

Our team’s logo and the style of our wiki had been settled.

### Saturday 25/7

Part of our team members took apart in a professional forum about prevention and rehabilitation of cardiovascular disease. The forum hold in Guangzhou, and some of the brightest experts converge at these forum. Such as Professor Hu Dayi who is one of the most famous cardiovascular disease experts and medical educators. We were very happy to be invited to participate in this forum. Thanks for organizers. We learnt a lot from the forum.

![](https://static.igem.org/mediawiki/2015/d/d2/SCUT-China-notebook-7-25.jpeg)

### Thursday 30/7

With the help of our senior fellow-student brothers and sisters, we studied the methods of Elisa and RNA extraction. We learned a lot in the course of experiments. But we still need to do the experiments by ourselves so that we can grasp the methods thoroughly.

### Friday 31/7

Today we went to the laboratory. We succeeded in reverse transcription of RNA and kept the cDNA in refrigerator. And our cell’s growth situation was favorable.

### Sunday 2/8

Today we reported our progress to our tutor in the meeting. After the meeting, we entered the cells room to continue our experiment. Cells were successfully subcultured by us.

### Monday 3/8

Today, in preparation, we prepared the solution before the experiment. We used cells which were available. First of all, we extracted the RNA from cells. After running the gel electrophoresis, we detected RNA’s concentration by ultraviolet spectrophotometer. And then, we synthesized cDNA by reverse transcription. Afterwards, we detected cDNA’s concentration, and we processed the data. And then we calculated the consumption of the reagents such as water, primers and PowerUp SYBR Green Master Mix. We expanded the cDNA with PCR. We used the cDNA which had been expanded to run the gel electrophoresis. After all the work had been done, we cleaned up the laboratory furniture.

On the other side, the work of culturing cells was well. There were more and more cells in our cells room.

![]()

### Tuesday 4/8

The result of experiment we had done yesterday was less than satisfactory. So we analyzed the reason why we failed. First of all, we found the mistake maybe caused from gene expression. Review of the literature revealed that PDE5A, GUCY1A3 and GUCY1B3 were expressed in kidney. The cells we used in experiment were HEK293. But we don’t know whether the genes we needed would express in HEK293 or not. So we had to consult with our tutor.

### Wednesday 5/8

Today we raised sample’s concentration to run the gel electrophoresis in the final step. Joy mingling with surprise, we found that both PDE5A and GUCY1B3 were expressed in the cells. DNA strips stained by GV could be seen clearly.

### Friday 7/8

Today we tested two methods of extract cGMP. The two methods were repetitive freeze-thawing and lysate. From the test result, we found that repetitive freeze-thawing extracting cGMP was more effective. And we also used sodium nitroprusside to treat cells but it cannot enhance the cGMP concentration.

![](https://static.igem.org/mediawiki/2015/2/28/SCUT-China-notebook-8-7.jpeg)

### Saturday 8/8

Today we invited SKLBC-China and SKLBC-GDSYZX teams to sharing academic experience. And we began a two-day cultivation today. We have the following arrangement today: extracting the plasmid, enzyme digestion and gel electrophoresis. Gel electrophoresis’s learning includes that how to make up the gel and how to recycle the gel. It’s our pleasure to share our experiment experience to members of high school team. We spent a beautiful day together.

### Sunday 9/8

We taught SKLBC-China and SKLBC-GDSYZX teams that transformation and coating plates. At last we exchanged our ideas about projects and gave some guidance to high school students. The two-day cultivation ended in a satisfactory way.

### Monday 10/8

We redesigned the αsubunit primers. We did the experiment again to find out the optimal primer. We successfully found out the best primer by summing up the experience of previous mistakes. Everything goes smoothly today.

### Thursday 13/8

We learned to use RT-PCR instrument from professor Ye Yanrui. The professor taught us seriously. We were grateful for him. At last, We put knowledge to practical usage. We set the PT-PCR condition first. And then put the solution into the instrument and start to run. After two hours waiting, the expression of gene had been detected. According to the diagram from the computer, we found that the results of three sample which were silenced gene PDE5A were satisfactory.

On the other side, we did the experiment of cell transfection, and we decided to evaluate the efficiency for eukaryotic cell transfection by using GFP as a report gene in common transfection ways.

![](https://static.igem.org/mediawiki/2015/e/ed/SCUT-China-notebook-8-13.jpeg)

### Friday 14/8

We reported the effect of gene silence to our teacher. We found out some abnormal data in the diagram. Maybe we had some mistake in the step of mixing the solution. So we prepared to do the RT-PCR once again. But this time, unfortunately, we put PCR Tubes Strip in wrong way. So we plan to do the experiment again tomorrow. We believed that failure can let us go further.

We observed a strong fluorescence from the cells transfected yesterday.

![](https://static.igem.org/mediawiki/2015/1/14/SCUT-China-notebook-8-14.png)

### Wednesday 19/8

We went to Traditional Chinese Medicine Hospital of Guangdong Province for visiting patients and making a survey. We communicate myocardial ischemia with the patients. We prepared some questions to ask them and we hope to get some useful references about the disease. We asked in detail about the disease and encourage them to establish confidence, adherence to treatment. On the one hand, in this interview, some patients pointed out that the frequency of taking medicine quite bothered him, and almost every patient should take with the emergency treatment drug all the time. We found that most of the patients not only had cardiovascular disease but also had diabetes mellitus, hypertensive disease and so on. On the other hand, we know that the healthcare system of China were good but quite inadequate. It definitely reduced patients’ financial burden. But we found that there were still some problems in the healthcare system. We concluded four major problems, for example, low coverage, lack of unity, the high insurance beyond reality and a waste of personal account in the process of constructing the social medical insurance system. After the visit, we shook hand with patients and showed our gratitude to the doctors and nurses who gave us support.

In the progress of experiment, we detected the concentration of cGMP in the cells which we transfected the day before yesterday by ELISA. And we found that the content of cGMP in the third group increased by twenty percent.

![](https://static.igem.org/mediawiki/2015/4/46/SCUT-China-notebook-8-19.jpeg)

### Thursday 20/8

We inoculated the shRNA puncture bacteria in different sites, the concentration of AMP in medium was 100ng/ul.

### Friday 28/8

Today we conducted the experiment of No’s positive control. We measured the concentration of cGMP by Elisa. There was a significant difference between adding cysteine 20min later and adding cysteine 30 min later.

![](https://static.igem.org/mediawiki/2015/f/f9/SCUT-China-notebook-8-28.png)

### Sunday 30/8

In preparation, we prepared the solution before the experiment. We used cells which were available. First of all, we extracted the RNA from cells which were over expressed βsubunit. And then we ran the gel electrophoresis. The plastic figure was clear and good. After that, we detected RNA’s concentration by ultraviolet spectrophotometer. At last we chose the best one in three samples. We conserved the sample for reverse transcription tomorrow.

### Monday 31/8

We extracted the RNA from cells which were used different concentration of virus to transfect the cell to silence gene. The concentration gradients of virus were 4μl, 8μl, 12μl, 16μl, 20μl and control. And then we ran the gel electrophoresis. The plastic figure was less than satisfactory.

![](https://static.igem.org/mediawiki/2015/0/0b/SCUT-China-notebook-8-31.jpeg)

### Tuesday 1/9

Today we screened the cells which had been stable transfected. And measured the fluorescence.

### Wednesday 2/9

We measured the concentration of sGC by Elisa today. The result is exciting. The alpha+beta group was significantly higher than the control group.

On the other side, we wanted to find out optimum concentration of cDNA which was added to PCR.

![](https://static.igem.org/mediawiki/2015/d/dd/SCUT-China-notebook-9-2.png)

### Thursday 3/9

We extracted the RNA from cells which were over expressed bothαsubunit andβsubunit. And we used the RNA to do reversed transcription. And we used those samples to do general PCR method. We ran the gel electrophoresis in the final step. We found that the expression ofαsub unit increased significantly. But, unfortunately, the expression of βsubunit was required to confirm since its control group is not available. To our surprise, the group which was added Nuclease-free water and primers appeared strip. We thought that it must have something wrong in our experiment today and we needed to find out the mistake.

![](https://static.igem.org/mediawiki/2015/2/23/SCUT-China-notebook-9-6.png)

### Friday 4/9

We chose the best primers which we designed the day before yesterday. We added different concentration of sample and found the best concentration in the experiment.

### Saturday 5/9

Today we redid the experiment on Friday. But we did the real-time PCR this time. We extracted the RNA from cells which were over expressed bothαsubunit andβsubunit. And we used the RNA to do reversed transcription. And we used those samples to do real-time PCR method. And then we ran the gel electrophoresis. The result made us disappointed. The group which added Nuclease-free water and primers appeared strip again.

![](https://static.igem.org/mediawiki/2015/9/99/SCUT-China-notebook-9-5.png)

### Sunday 6/9

We tried again today. We got excited by the result. The plastic figure showed that the over expression of αsubunit andβsubunit was effective. But the effect of shRNA was not obvious.

![](https://static.igem.org/mediawiki/2015/d/d1/SCUT-China-notebook-9-3.png)

### Monday 7/9

Up to now, we finished collecting all the data we need. We further processed of our data.

### Friday 11/9

We flung ourselves to our wiki.

### Friday 18/9

We got everything done today Ahahahahaha~~~