1. Growth curve of E.coli containing part K577881 in different concentration of arsenite (arsenite) 

Fig.1 showed that the growth of bacteria was not affected in the range of 5ppb-10ppm of arsenite solution (As³⁺). However, as the concentration of arsenite increased to 50ppm, the growth of the bacteria was seriously inhibited, suggesting the toxictity to cells at this level.

Fig.1

Fig.1 Growth curve of E.coli in different concentration of arsenic (As³⁺). Constructed plasmid pSB1C3-pArsR-GFP containing J33201 part was transformed into E.coli DH5α strain. Single colony was selected to inoculate LB broth containing chloramphenicol and cultured overnight. Then overnight culture was inoculated in the fresh LB medium containing chloramphenicol 34μg/ml at a ratio of 1:100, mixed well and divide into tubes. Different concentrations of arsenite(As³⁺) solutions were added into the test tubes, respectively, so that the final concentration of arsenic was (0, 5ppb, 10ppb, 50ppb, 100ppb, 500ppb, 1ppm, 5ppm, 10ppm, 50ppm and 100ppm). Samples were taken at different time points of 0h, 1h, 2h…8h and overnight (16h) at one hour interval and OD600 value were measured with a Multiskan Spectrum Microplate Reader.

2. GFP expression of E.coli under the control of J33201 induced by different concentration of arsenite (As³⁺)

As shown in Fig.2, our constructed biosensor based on J33201 part is very sensitive to arsenite(As³⁺) and the fluoresence signal can be detected at 50ppb arsenite. Significant dose-dependent effect was observed at the range of 50ppb-1ppm arsenite. The threshold of this biosensor is 10ppm. As the arsenite concentration increased to 50ppm, it does’t work due to the toxicity to cell at this level.

Fig.2a

Fig.2b

Fig.2 GFP expression of E.coli under the control of J33201 in different concentration of arsenite (As³⁺). (a) Overnight cultured bacterial solution was inoculated in LB broth containing chloramphenicol at 1:50 to expand the culture, and the experiment was started when OD600 reached 0.4-0.6. Different concentrations of arsenite solutions were added into the test tube, respectively, so that the final concentration of arsenic was (0,5ppb, 10ppb, 50ppb, 100ppb, 500ppb, 1ppm, 5ppm, 10ppm, 50ppm, 100ppm). Samples were taken at overnight (16h). GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time.  (b)The bacteria were centrifuged, and the pellets were was observed and photographed under Blue Light Gel Imager.

As it is shown in Fig.3, our constructed biosensor by J33201 reacted rapidly in arsenite solution. When the arsenite is added for 4 hours, the corresponding fluorescence signal can be detected at 100ppb. As time increased, weak signal can be detected at 50ppb, indicating that the biosensor can work within 4 hours.

Fig.3 GFP expression of E.coli in different concentration of arsenite (As3+) at different time points under the control of J33201. Overnight cultured bacterial solution was inoculated in LB broth containing chloramphenicol resistance at 1:50 to expand the culture, and the experiment was started when OD600 reached 0.4-0.6. Different concentrations of arsenite solutions were added into the test tube, respectively, so that the final concentration of arsenic was (0, 5ppb, 10ppb, 50ppb, 100ppb, 500ppb, 1ppm, 5ppm, 10ppm, 50ppm, 100ppm). Samples were taken at different time points of 0h, 2h, 4h, 6h and overnight (16h). GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time

To summary, our biosensor constructed on the basis of J33201 is sensitive and fast. The biosensor can detect 100ppb-10ppm arsenite (As³⁺) in water within 4 hours, and the minimum detection limit can reach 50ppb with the extension of time.