We constructed our NO sensor in the pCDFDuet-1 plasmid (Figure 1)
According to the results of the ShanghaiTechChina_B 2016 team, 100μM SNP aqueous solution can continuously release NO, and the NO concentration is stable at about 5.5μM. This concentration is the concentration of NO in patients with IBD. Therefore, in the subsequent test of the NO sensor, the final concentration of the SNP aqueous solution we added was 100 μM.
We transformed the constructed NO sensor plasmid into DH5α, and cultured at 37 ℃ overnight to dilute to OD = 0.2. After 1.5 h of growth at 37 ℃, the inducer IPTG and SNP aqueous solution were added. After 6 h at 37 ℃, 1 mL of the bacterial solution was centrifuged at 8000 rpm for 1 min (Figure 2).
From left to right: control, 0.5mM IPTG without SNP, 1mM IPTG without SNP, 0.5mM IPTG with 100μM SNP, 1mM IPTG with 100μM SNP.
We also tested the response of BBa_K381001 to NO in the Distribution Kit (Figure 3).
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 4).
A, Histogram of GFP fluorescence: LB control, without SNP, with 100μM SNP. B, GFP fluorescence image from top to bottom: without SNP, with 100μM SNP.
At the beginning of the experiment, we directly constructed the plasmids of YebF-IL10 and YebF-myrosinase, but the effect of extracellular detection was not satisfactory. During the participation in CCiC, iGEMer suggested that we first start from testing the performance of the secretory tag, so we constructed the plasmid YebF-GFP shown in Figure 5A. To express IL10 and myrosinase, we constructed plasmids IL10-flag and myrosinase-his as shown in Figure 5B and Figure 5C.
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℃.
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.