Team:Shanghai HS United/Collaborations
We have interacted with the Nanjing_High_School team throughout the iGEM. Although we have distinct topics, we exchanged our opinions on how to proceed our projects. On July 9th, we attended a face-to-face meet-up at Fudan University, Shanghai. It was a great honor that Dr. Rongwei Chu, an associate professor at School of Management, Fudan University, helped us with hosting the meet-up event. Dr. Chu led us to discuss crucial issues on fundraising and expert interview. Since Nanjing_High_School started their human practice events earlier than we did, they shared their experience with us. In detail, they helped us with figuring out the meaning of expert interview. “It will be great for you to filter experts’ opinions related with your research topic since sometimes expert may mention a lot informaiton, some of which may not directly with wha you are doing about.” Nanjing_High_School told us that. In addition, time arrangement was another issue that they had reminded us of. It will not be a good idea to interview over 3 experts in one day, especially when these experts worked in different places. It will be better to leave sufficient time for every expert interview, considering transportation time and interview length. It was also a precious piece of suggestion for us.
Construction and measurement the expression of GFP in the construction of type IIS. (Collaboration with Xiamen_City)
Meanwhile, since both of us were preparing grand promotion event in the coming days., We discussed two major concerns about this: Whaf elements should we include in launching the events? How should we do with the charity fund if we manage to get some during the events? After a fierce discussion, we reached an agreement that both of us may hold integrated talent show in related with our own research topic and we may try to find some research institute to donate the fund we may get from the promotion events to support the sustainability of the related research .
Material: pSB1K3-LacZ, pSB1K3-GFP, pSB1K3-RBS-A-GFP, pSB1K3-RBS-B-GFP, pSB1K3-RBS-D-GFP, MG1655 bpul, DH10B bpul, BL21(DE3) bpul, loading solution (6 μL), deionized water (1000 μL), marker solution, Coomassie brilliant blue dye.
Note: pSB1K3-LacZ, pSB1K3-GFP, pSB1K3-RBS-A-GFP, pSB1K3-RBS-B-GFP, pSB1K3-RBS-D-GFP are used for collaboration with the team Shanghai_HS_United, and these plasmids were expression in strain E. coli DH10B.
1.Add 24 μL pSB1K3-LacZ, pSB1K3-GFP, pSB1K3-RBS-A-GFP, pSB1K3-RBS-B-GFP, pSB1K3-RBS-D-GFP, MG1655 bpul, DH10B bpul, BL21(DE3) bpul to 1.5mL tube respectively, and then add 6 μL loading.
2.Add 500 μL deionized water and centrifuge the tubes respectively.
3.Add another 500 μL deionized water, blow and suspend the solution.
4.Put the tubes 99℃ water for 15 minutes.
5.After heating, centrifuge the tubes for 5min.
6.Take and install the gel, add the electrophoresis buffer to the sample hole, and check if there is any leakage. Add marker, to the first and last sample holes, add pSB1K3-LacZ,pSB1K3-GFP and pSB1K3-RBS-A-GFP, pSB1K3-RBS-B-GFP, pSB1K3-RBS-D-GFP, MG1655 bpul, DH10B bpul, BL21(DE3) bpul to the other sample holes, respectively.
7.Connect the electrophoresis device to the power supply. Connect the positive electrode to the tank and the negative electrode to the slot for electrophoresis. The voltage is adjusted to 160V.
8.Turn off the power supply and disconnect the electrode until the bromophenol blue reaches the bottom of the release adhesive. Remove the glass sheet from the electrophoresis device and then remove the gel.
9.Soak the gel in Coomassie brilliant blue dye and dye it in a horizontal shaking bed for 15 min.
Observe the protein bands
Material: LB medium (2mL), pSB1K3-RBS-A-GFP, pSB1K3-RBS-B-GFP, pSB1K3-RBS-D-GFP, pSB1K3-GFP, pSB1K3-FnCas12a, pSB1K3-LacZ, pSB1K3-J23100-B0034-ter
1.Take several test tubes and add to 2mL KAN medium respectively.
2.Take the cultured pSB1K3-RBS-A-GFP, pSB1K3-RBS-B-GFP, pSB1K3-RBS-D-GFP, pSB1K3-GFP, pSB1K3-Fncas12a, pSB1K3-LacZ, pSB1K3-J23100-B0034-ter, use the needle tip to take a small amount, and put it onto the shaking table.
3.Measure the Fluorescence value and the OD value before and after centrifugation.
Fig. Fluorescence /OD value before and after centrifugation
The results showed that BBa_B0034 RBS in type IIS assembly has low expression efficiency, and the RBS-A was the best.