Difference between revisions of "Team:NEU China A/Improve"

This year, we chose to use BBa_K381001 (PyeaR-GFP) as an alternative to our inflammatory sensor. The promoter PyeaR is sensitive to nitrate and nitrite. When nitrate and nitrite enter E. coli, they are converted to nitric oxide. Nitric oxide binds to the repressor protein NsrR, which inactivates PyeaR to inhibit transcription of downstream genes.

We learned that iGEM 2010 Team BCCS-Bristol used BBa_K381001 to detect nitrate and nitrite in the soil. The content of nitrate and nitrite in the soil can reflect the fertility of the soil. Farmers can determine which soils are fertile by detecting the fluorescence of GFP. In this way, farmers only need to apply fertilizer in places where there is no fertility, which can save excess fertilizer. Given the economic costs and the impact of eutrophication on ecosystems, the use of BBa_K381001 has great benefits for both farmers and the environment. However, due to the influence of outdoor temperature, GFP fluorescence fluctuates greatly. This instability is unfavorable for the detection of soil fertility. In addition, the detection of GFP fluorescence requires special equipment that is not readily available to farmers. Therefore, we replaced GFP with blue chromoprotein amilCP for visual detection of soil fertility. On the one hand, amilCP expression is less affected by temperature and is a more stable reporter than GFP. On the other hand, amilCP does not require special equipment to be visible to the naked eye. Therefore, we believe that our improved part BBa_K2817007 is very beneficial to farmers.

According to the results of theShanghaiTechChina_B 2016 team, 100μM SNP aqueous solution can continuously release NO, and the NO concentration is stable at about 5.5μM. Since our project also tested for inflammatory signals, we chose this concentration before testing for BBa_K381001 and BBa_K2817007.

The construction of BBa_K381001 can be seen from Figure 1A. We transformed the plasmid containing BBa_K381001 into DH5α, and cultured at 37 ℃ overnight to dilute to OD = 0.4. Then we took half as control and the other half added SNP aqueous solution and induced at 37 ℃ for 6 h. Then we detected the fluorescence using a microplate reader and a fluorescence microscope (Figure 1B, 1C). We can see that PyeaR can be effectively activated by NO with almost no leakage.

Figure 1. The test of BBa_K381001. A, the construction of BBa_K381001. B, Histogram of GFP fluorescence: LB control, without SNP, with 100μM SNP. C, GFP fluorescence image from top to bottom: without SNP, with 100μM SNP.

The construction of BBa_K2817007 can be seen from Figure 2A. We transformed the plasmid containing BBa_K2817007 into DH5α, and cultured at 37 ℃ overnight to dilute to OD = 0.4. Then we took half as control and the other half added SNP aqueous solution and induced at 37 ℃ for 6 h. We also set up negative control group which doesn’t contain amilCP. After 6 h at 37 ℃, 1 mL of the bacterial solution was centrifuged at 8000 rpm for 1 min (Figure 2B). We can see the result of PyeaR being activated by NO by the naked eye.

A, the construction of our YebF-GFP using strong promoter. B, the construction of IL10 production module. C, the construction of myrosinase production module.

When testing the validity of the secretory tag YebF, we transformed the constructed YebF-GFP plasmid into DH5α and cultured overnight at 37 ℃. The supernatant was centrifuged at 3000 rpm for 5 min, and the supernatant was taken for fluorescence detection. The results are shown in the Figure 6. But we forgot to set up a positive control group. Due to time constraints, we have no time to repeat this experiment.

We transformed the IL10-flag plasmid into BL21, and incubated at 37 ℃ overnight to dilute to OD = 0.2. After 2 h of growth at 37 ℃, IPTG was added and induced at 30 ℃ for 16 h. Then, the bacterial solution was lysed and the expression of IL10 was detected by western blot (Figure 7).

Lane 1: Negative control (cell lysate without IPTG induction); Lane 2: 0.5mM IPTG, Lane3: 1mM IPTG induction for 16h at 30℃.

We transformed the plasmid of myrosinase-his into BL21, and cultured at 37 ℃ overnight and diluted to OD = 0.2. After growth for 2 h at 37 ℃, different concentrations of IPTG were added and induced at 16 ℃ for 16 h. The bacterial cell lysis was then performed to detect the expression of myrosinase by SDS-PAGE (Figure 8).

Lane 1: Negative control (cell lysate without IPTG induction); Lane 2: 0.25mM IPTG; Lane3: 0.5mM IPTG, Lane4: 0.75mM IPTG induction for 16h at 16℃.

3.Kill Switch

We inserted the reporter gene amilCP into the pColdI plasmid to characterize the performance of the cold shock promoter PcspA (Figure 9A). We inserted maz-F into the pColdI plasmid to build our kill switch (Figure 9B).

A, the construction of PcspA-amilCP plasmid. B, the construction of PcspA-mazF plasmid.

We transformed the PcspA-amilCP plasmid into DH5α and cultured overnight at 37℃. The overnight culture was diluted to OD = 0.2 and allowed to grow for 2 h at 37℃. It was then divided into different concentrations of IPTG at 16℃ and 37℃ for 6 h (Figure 10).

From left to right: 37℃ without IPTG, 37℃ with 0.5mM IPTG, 37℃ with 1mM IPTG, 16℃ without IPTG, 16℃ with 0.5mM IPTG, 16℃ with 1mM IPTG.

We transformed the constructed PcspA-mazF plasmid into BL21, added 1 mM IPTG to the plate, and cultured at 16℃ for 16 h (Figure 11A). We then cultured BL21 transformed with the PcspA-mazF plasmid overnight at 16℃. After diluting to OD=0.2 on the next day, the cells were cultured at 16℃, and the OD value was measured every hour for 9 hours (Figure 11B).

A, the plate of BL21 with and without killer gene under induction. B, the growth curve of BL21 with and without killer gene under induction.