Latest revision as of 16:18, 21 October 2020

E. coli chromosomal ars promoter with arsR repressor gene

This part consists of the promoter of the E. coli JM109 chromosomal arsenic detoxification operon (ars operon), including the ArsR repressor binding site and the arsR gene encoding the arsR repressor protein, together with its ribosome binding site. Addition of any other genes to the 3' end of this part will result in their expression being dependent on the presence of sodium arsenate or sodium arsenite. Arsenite or arsenite anion binds to the repressor protein ArsR, resulting in inability to repress the promoter. Based on our experiments, a concentration of 1 micromolar sodium arsenate in LB is sufficient for essentially full expression, though this will vary according to conditions.

Sequence and Features

Supplementary material offered by other groups

The following is from TJU-China 2018. We used the part:Bba-J33201 saved in plate3 and wanted to build a new arsenic induction loop through it. Therefore, we first added 10μlddH2O to the corresponding hole, and then took PCR was performed at 1μl, and the corresponding DNA fragment was obtained.

Figure1. The result of nucleic acid gel electrophoresis of Bba-J33201 after PCR. Lane M, Marker. Lane 1-6，Bba-J33201

Then we performed PCR on the promoter fragment and ArsR fragment in this fragment, and the results are as follows.

Figure2. The result of nucleic acid gel electrophoresis of Ars Promoter after PCR. lane M, Marker. Lane 1-4, Ars Promoter.

Figure 3. The result of nucleic acid gel electrophoresis of ArsR Protein after PCR. Lane M, Marker. Line1, ArsR Protein.

Characterization

Worldshaper-Wuhan 2019's Characterization

Detail information please check our wiki website

http://2019.igem.org/Team:Worldshaper-Wuhan-A/Measurement

Aim of experiment

Based on J33201 part, a biosensor pSB1C3-pArsR-RBS-GFP(K3153000) was constructed to detect arsenite(As3+) in water and contributed to this well-characterized part.

Methods

1. Growth curve of E.coli containing part J33201 in different concentrations of arsenite(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 34 μg/ml chloramphenicol at a ratio of 1:100, mixed well and divided into tubes. Different concentrations of arsenite (As3+) solutions were added into the test tubes, respectively. The final concentration of arsenic was 0, 5 ppb, 10 ppb, 50 ppb, 100 ppb, 500 ppb, 1 ppm, 5 ppm, 10 ppm, 50 ppm and 100 ppm. Samples were collected at different time points of 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h and 16 h. OD600 values were measured by the Multiskan Spectrum Microplate Reader.

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

Overnight cultured bacterial solution was inoculated in LB broth containing chloramphenicol at 1:50 to expand the culture. When OD600 reached 0.4-0.6, experiments were performed . Different concentrations of arsenite were added into the test tube, respectively. The final concentration of arsenic was 0, 5 ppb, 10 ppb, 50 ppb, 100 ppb, 500 ppb, 1 ppm, 5 ppm, 10 ppm, 50 ppm, 100 ppm. (1) Samples were collected at 16 h. GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time. The bacteria were centrifuged, and the pellets were was observed and photographed under Blue Light Gel Imager. (2)Samples were collected at different time points of 0 h, 2 h, 4 h, 6 h and 16 h. GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time.

Results

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

Fig.1 Growth curve of E.coli in different concentration of arsenic (As3+).

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

(Fig.2a)

(Fig.2b)

Fig.2 GFP expression of E.coli under the control of J33201 in different concentrations of arsenite (As3+).

As shown in Fig.3, our biosensor by J33201 reacted rapidly in arsenite solution. After 4 h following arsenite treated, the corresponding fluorescence signal was detected at 100 ppb. As time increases, a 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.

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. With the extension of time, the minimum detection limit can reach 50ppb.

Reference:

Anal Bioanal Chem. 2011 May;400(4):1031-9. Epub 2011 Mar 27.

Characterization

Team Groningen 2020's Characterization

Figure 2. mCherry expression under the control of BBa_J33201 measured as Fluorescence/D600 at various concentrations of Arsenate (As(V)) at different timepoints up to 4 h.

Figure 3. mCherry expression under the control of BBa_J33201 measured as Fluorescence/D600 at various concentrations of Arsenate (As(V)) at different timepoints up to 21h.

Figure 4. Growth curve of the E. coli strains carrying the parsR-mCherry fusion construct at D600 at various concentrations of Arsenate (As(V)) at different timepoints up to 21h.

Team Groningen 2020 has improved the characterisation of the BioBrick BBa_J33201, which is based on the ArsR transcriptional repressor system of the arsenic detoxification operon (arsR-operon) in E. coli (Xu et al., 1995). The biobrick includes the ArsR repressor binding site, the arsR promoter, a ribosomal binding site and the arsR gene encoding the ArsR repressor protein.

Under standard conditions, the ArsR protein regulates its own expression, inhibiting its own promoter (Diorio et al., 1995). When an arsenic ion is present, it will bind to ArsR. This complex is not able to inhibit the promoter, making expression of upstream reporter genes possible.

Figure1. Construct overview: mCherry gene (BBa_J06602) under the control of the BBa_J33201 ArsR promoter with addition of two stop codons (BBa_B0015) in backbone BBa_J04450.

For our characterization of the sensitivity of ArsR to As(V), we constructed a fluorescent As(V) detection module. In a first cloning step, BBa_J33201 was cloned upstream of BBa_J06602 which contains a ribosomal binding site and the structural gene for the red fluorescent gene mCherry (see Fig 1) in plasmid backbone BBa_J04450. In a second cloning step, the double transcriptional terminator BBa_B0015 was cloned downstream of BBa_J06602 into the plasmid backbone BBa_J04450. All cloning steps followed the 3A assembly standard. The transformations were performed with chemically competent E.coli Top10 cells via heat-shock transformation. Cells were grown in LB-medium at 37°C supplemented with 100 μg/ml ampicillin and 50 μg/ml kanamycin, respectively, for plasmid selection (final concentrations). Uptake of the plasmid was confirmed using colony PCR.

To assess the functionality of the As(V) detection module, different concentrations of arsenate, As(V), were added to the E. coli cells in a 96 well plate (Vfinal =200μl). The final As(V) concentration in the wells was 0.1ppm, 0.1ppm, 1ppm, 10ppm or 100ppm, respectively. mCherry fluorescence (587nm excitation/ 610nm emission) and OD600 were measured at ten different time points (0h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h and 24h) using a Microplate reader. The results of our experiments are shown in the graphs below.

By looking at the mCherry fluorescence signal, it can be concluded that the expression of mCherry is As(V) dose-dependent (Figure 2 and 3). We recommend a concentration of 10ppm for an optimal fluorescence/OD600. We did not observe the toxic effect at high concentrations of the arsenic ion described by Team Wuhan 2019, (Figure 4). After 21h incubation at As(V) concentrations of 50ppm and 100ppm we did not observe a decrease in OD600, which was observed by Team Wuhan 2019. This could be due to our team using As(V) while Team Wuhan was As(III) for their analysis. We did not identify any As(V) in a local soil sample.