Team:Xiamen City/Collaborations
Collaboration with IGEM team Shanghai-High_School
In August, we made a long-term collaboration and communication with an iGEM team (Shanghai_High_School). This team’s purpose is to design a color indicator based on the mechanism of RNA thermometers for vaccines using TXTL Cell-Free Expression method to ensure the preservation of vaccines during transportation. RNA thermometer is a temperature-sensitive non-coding RNA molecule which regulates gene expression.
We exchanged our detailed research information and shared insights on specific experiments with each other. Moreover, we shared our laboratories with them, giving them instructions on how to use our equipment. In return, they helped us to make M9 culture media. They also discussed the mini program like how to make better content with us and promised to promote it to their friends.
Construction and measurement of the expression of GFP of type IIS
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.
10. Observe the protein bands
culture
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-Facas12a, 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.
We constructed the secretion of proteins by constructing signal peptides plasmid (GFP). (Collaboration with Shanghai_City)
Measure the fluorescence secretion of E. coli
Methods:
1.20 bacteria culture 20 ul were absorbed and put into the microtitration plate.2.Entrifuge the remaining bacteria solution at 12000g for 1 min.
3.Draw 20ul of supernatant from 20 bottles after centrifugation and put it into the microtitration plate.
4.Put the plate into the instrument to measure the absorbance and fluorescence.
Results:
Fig1. The concentration of microbes in suspensions and supernatants
Fig2. The fluorescence of GFP in suspensions
Fig3. The fluorescence of GFP in supernatants
As can be seen from Fig1, the absorbance of the bacterial solution is generally higher than that of the supernatant, while the fluorescence measurement shows that the value of the supernatant is relatively small, generally 30-40, compared with the value of about 1000 of the bacterial solution(Fig2 and Fig3). The fluorescence of pelB-GFP was higher than the control. It turned out that the protein pelB-GFP was expressed successfully and secreted to the extracellular environment.
the low fluorescence of supernatant indicates that E. coli can only secrete a small amount of fluorescent substances to the extracellular environment. And liken to the control group, it is easy to confirm the specific amount of fluorescent substances secreted.
Next Day
Methods:
1. Take out the E. coli cultured in a shaker yesterday (6 in total) and divide them into three groups: no GFP gene, GFP gene without signal peptide and GFP gene with signal peptide. Proper amount of bacterial suspensions and supernatant samples after centrifugation were successively extracted for absorbance detection.
2. Extract appropriate amount of bacteria solution: extract 200 microliters of bacteria solution from each tube and put it into group A 1-6 Wells of 96-well plate
3. Supernatant extraction: again, extract 500 microliters of bacterial liquid from each tube, place them in the labeled EP tube, and put them into a centrifuge for centrifugation. After centrifugation, 200 microliters of supernatant were extracted from each tube and put into group B Wells 1-6 of the 96-well plate.
4. Put the 96-well plates into the microplate reader and test their absorbance.
Results:
Fig4. The fluorescence of GFP in suspensions and supernatants
In suspension, GFP containing E. coli have high value of fluorescence, while adding signal peptide has a little effect. So the signal peptide can improve the absorbance a little.
Idetification of bacteria from aniline-polluted sludge
Friendly Exchange with Cyanobarrier
At 4pm on July 27th, we had a friendly exchange with Cyanobarrier (Official Team Name: Shanghai_HS) from Shanghai through WeChat online video. Our research institute and the team from Shanghai exchanged and Shared their experience in biology, economics and environmental protection.
Through our understanding, Cyanobarrier focuses on cyanobacterial toxins in the hope that through the application of a new type of enzyme will loop structure, previous methods in their research, whether it is through the use of physical degradation of activated carbon, or is the degradation of chemically are difficult to clear the annular cyanobacterial toxins in the stability of the structure of the clean. So they plans to biodegrade it using a new enzyme to break down the ring-shaped cyanobacteria toxins into chains, which in turn divide the chains into smaller pieces, making it easier to break down.
In this exchange, we shared the progress and efforts we had made to realize and complete our project, such as some models of social research, field visits and interviews. We also had a general understanding of each other's projects. It has defined the goal and direction of our research on the way forward.
In addition, we also Shared the Logo design with each other. The Cyanobarrier’s logo has a hand with a drop of water to keep the cyanobacteria toxin out, meaning it wants to keep the cyanobacteria toxin out of the natural environment to protect the water and human society. We take egrets as the main body, and the blue and white tone expresses our determination and persistence in dealing with water pollution.