The Collaboration with Keystone
Afternoon: Induced expression of E.coli
Selecting the fungus (dipped with a white gun tip). The spoons still remained in the nutrient medium
Multiply the fungus solution into 500-1000ml LB medium (concentration ratio 1:100) (500mL LB was taken, which is 5mL fungus solution)
Cultivate for 4 hours (for E.coli, the appropriate temperature is 37°C; Yeast at 30°C)
When the OD600 reaches 0.6-0.8, the induced expression is needed to be done. (Under 600 nanometer wave length, the optical density should be 0.6-0.8)
Lower the temperature to 16-20 degree, add the IPTG inducer. The concentration should be 100μM (100μ mol/L).
Wait for a night
Keystone collaboration SDS-page of running protein gel (not finished) (Summary error: red and black line were inserted in the opposite direction, so the gel was ran in the opposite direction) Run the gel:
1.Wash all the equipment
2.2.Dilute the prefabricated glue with TRIS-Hydrochloric acid buffer.
3.The (LCC) proteins were added to the four PCR tubes. The LCC proteins in the tubes were 70, 50, 25 and 15 microlitre (mixed at different concentrations to 70μL)(The control group was supposed to be needed, and one group was induced and the other was not, but Keystone didn’t make the inducer, so we can merely use the gradient to compare)
4.Add hydrochloric acid buffer to each PCR tube (actually I'm not sure if I added hydrochloric acid buffer when I looked back at this record, but double check), so that the liquid in the PCR tube is 70 microliters.
5. Add the liquid from the PCR eight-tube into the PCR tube with a lid.
6. Add the protein loading buffer into the PCR tube, and the ratio of the loading buffer to the protein concentration is 1:5 (so take 10 L loading buffer into 50 L protein).
7. In a centrifuge, centrifuge the liquid on the tube wall to the bottom of the tube
8. Heat proteins in a water bath until heated to 70%
9. Remove the seal under the configured meringue (purchased) and secure it to a rack and cover to seal it.
10. Dilute the SDS buffer (20ml) with water and the ratio of buffer to water is 1:4
11. Pull the comb out and add buffer. (Both positive and negative are need to be added). The one for negative side should be added between the high and low glass,
12. Add the sample to the electrophoresis gel
13. Set the voltage to 80V and wait
The collaboration with the Beijing No.4 middle school:
Help the fourth middle school to match and run the gel.
We the 101 iGEM team initiate and participate in multiple exhibition seminar. We have joined our effort in several online meetings with more than 30 iGEM teams around China, including high school teams and professional undergraduate teams.
1.In the meeting organized by QHFZ-China from Tsinghua University High School, we are the most influential team on presentation, and were remarked and appreciated by the invited expert judges.
2.We also participated in the meeting set up by Keystone and organized our own meeting for more than 20 iGEM teams in China. In all these meetings, we joint our efforts in making iGEM teams a big family, in which we encouraged each other and supported other teams generously.
3.Our team collaborated with Keystone Academy by joining the Synbio fair hosted by Keystone together with other seven schools in Beijing. We made a poster exhibiting our project and made a presentation about it. This collaboration experience builds good relationships between our two school students, and it also allows us to spread synthetic biology to more students, which has great educational meanings.
On August 1st, We participated in an online meeting of iGEM teams in Beijing and introduce our project to them, especially our future prospects and plans. For example, we want to do research on methods that can preserve the man-made breast milk well, set a low price that all family can afford, and add more proteins into it to make it more like the natural one
Above all, in HP practice, laboratory and research processes, we all did many collaboration.
In wet lab, we provided sufficient advises for QHFZ-China iGEM team, helped Beijing No.4 High School in deploying solutions and equipment for experiments.
In human practice, we supported Keystone in finding resources for background research and finished video game.
In exhibitions, we were working on giving presentation and got many feedback.
All we did is to contribute our own individual effort in making iGEM not only a competition, but a small world that support all the dreams and goals.
In June, the Keystone Academy held up an activity of making a video game. It is a passing-task game that bases on a big script. This script tells a story of a primary school student called Xue Shengwu (in Chinese, this name pronounces the same as “learning biology”) who turned small and had an adventure in the microworld. Along this way, he passed many tasks and learned a lot about synthetic biology.
These small tasks would be written by each school that participates in this collaboration. In each task, our main character Xue Shengwu will learn about one piece of knowledge about synthetic biology. There are 7 pieces of story in total: Central dogma, PCR technology, CRISPR, biobrick, Gibson technology, golden gate technology, and directed evolution.
We, BJ101HS from Beijing No.101 Middle School, wrote the “biobricks” portion. It is a technology that allows dozens of genes can be expressed by a single plasmid. For each gene that need to be expressed, one EcoRⅠ and one XbaⅠ binding sites are placed on one side of the gene, and one SpeⅠ and one PstⅠ binding sites are placed on the other side. One key knowledge is that the binding sites of SpeⅠ and XbaⅠ are the same, which made it possible for the broken sites of SpeⅠ and XbaⅠ to link together. For plasmid 1, the EcoRⅠ site and SpeⅠ site was cut and gene was extracted. And for plasmid 2, EcoRⅠ site and XbaⅠ site were cut. Then the SpeⅠ (from gene 1) & XbaⅠ (from gene 2) site are linked, and EcoRⅠ from gene 1 & PstⅠ from gene 2 are linked. In this way a new single plasmid is created, and this process can be repeated until the complex plasmid is created. In our story, Xue Shengwu woke up in a PCR machine. A restriction enzyme, Green, told him about this biobrick technology that is processing in the PCR machine. Finally, Green led Xue Shengwu to the “restriction enzyme training base” and asked Xue Shengwu to practice assembling a complex plasmid.
After we finished this part of the script, we also participated in drawing the animation of the video game. Our art team leader led her group members and learned about how to operate the drawing software. This is a meaningful activity which helped to spread the synthetic biology knowledges to more people.