iGEM 2019 — Team Worldshaper-Wuhan

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# Team:Worldshaper-Wuhan/Notebook

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# Notebook

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## Notebook

2019.7.20 Sat

Read the literature

2019.7.21 Sun

Read the literature on arsenic treatment in Bangladesh

2019.7.22 Mon

Upon first entry into the laboratory, arsenic adsorption plasmids had been synthesized by two plasmids, pEP-SpPCS and pET-orpF-fmt, transforming E. coli BL21(DE3) and culture overnight

2019.7.23 Tue

Select a single colony on the plate, convert the colonies of pEP-SpPCS and pET-orpF-fmt, inoculate LB broth containing ampicillin resistance, and culture overnight.

According to the designed primer, J33201fragment was amplified, electrophoresis identification, gel cutting and purification, enzyme cutting E/S, gel cutting and purification

2019.7.24 Wed

The bacterial liquid cultured overnight was re-inoculated into fresh LB broth containing ambenzyl resistance at a ratio of 1:100 for expanded culture and detection of OD600. When OD value was 0.6-0.8, IPTG(1mM) was added for induction expression and low temperature was induced overnight at 28℃.

2019.7.25 Thu

Protein samples were prepared with 5\* SDS-PAGE loading buffer

SDS-PAGE gel was prepared and SDS-PAGE electrophoresis was carried out on the prepared samples

After electrophoresis, Coomassie brilliant blue was dyed for 30min, then decolorized and washed overnight

2019.7.26 Fri

SDS-PAGE electrophoresis results were observed and photographed

Prepare HUST communication materials

SDS-PAGE adhesive was prepared and WB experiment was carried out. Upper sample, electrophoresis, membrane transfer, incubation of primary antibody, anti-Histag 1:10, 000,4 ℃ overnight

2019.7.27 Sat.

No experiments, participate in the HUST exchange

2019.7.28 Sun

rest

2019.7.29 Mon

SDS-PAGE adhesive was prepared and WB experiment was carried out. Upper sample, electrophoresis, membrane transfer, sealing, incubation of primary antibody, anti-Histag 1:10,000,

2019.7.30 Tue

Wash, incubate the secondary antibody, wash, expose, X-ray film, analyze the results and find that the signal is too strong, WB needs to be done again.

2019.7.31 Wed

Prepare for meetup in hangzhou. Experiment suspended

2019.8.1 Thu - 8.4. Sun

In HANGZHOU MEETUP

2019.8.5 Mon

SDS-PAGE adhesive was prepared and WB experiment was carried out.Upper sample, electrophoresis, membrane transfer, sealing, incubation of primary antibody, optimization of reaction conditions, anti-histag 1:100,000

2019.8.6 Tue

Wash, incubate the secondary antibody, wash, expose and photograph, and obtain satisfactory experimental results

2019.8.7 Wed

Plasmid containing GFP,( plate1,4K), and 10ulddH20 were found from iGEM distribution kit, and the GFP fragment was amplified as the template. Electrophoresis verification showed that the electrophoresis result was not ideal

2019.8.8 Thu

According to the redesign of primers, repcr, electrophoresis, gel cutting and purification

2019.8.9 Fri

IGEM distribution kit linearized pSB1C3 plasmid, enzyme digestion, E/P, electrophoresis validation, gel purification

J33201 fragment was cut and purified, E/S was cut and purified

The fragment of RBS-GFP was cut and purified, enzyme digestion, X/P, electrophoresis verification, and cut and purified

T4L ligase connected the above fragments at 16℃ for 1h

The junction product transformed E. coli DH5 a, coated with a plate containing chloramphenicol resistance, and cultured overnight

2019.8.10 Sat

Positive clones grew on the plate, and the plate was put in a 4℃ refrigerator for experiment on Monday

2019.8.11 Sun

Rest and do no experiments

2019.8.12 Mon

The recombinant j33201-rbs-gfp was verified by colony PCR on the plate, PCR products were electrophoresis, positive clones were selected, chloramphenicin-resistant LB broth was inoculated, and overnight culture was conducted

2019.8.13 Tue

The bacteria solution was reserved with glycerin bacteria, the remaining small plasmid was identified by enzyme digestion, and three plasmids were selected and sent to the company for sequencing

2019.8.14 Wed

Sequencing results were in line with expectations and functional measurements were initiated. Prepare arsenic preparation, prepare mother liquor

2019.8.15. Thu

Growth curve experiment

Induction curve experiment

2018.8.16 Fri

Analysis of the data

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# Team:Worldshaper-Wuhan/Experiments

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# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:Worldshaper-Wuhan/Results

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# Results

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## Results

# **Biosensor**

## **1.** **C****onstruction of plasmid pArsR-RBS-GFP (K3153000)**

##### 1) PCR results of J33201

We used the part J33201 from the iGEM distribution kit and aimed to design a new arsenic biosensor through it. So we first added 10μl ddH2O to the corresponding hole, and then took PCR was performed at 1μl, and the corresponding DNA fragment was obtained (Fig.1).

![Results(图1)](https://static.igem.org/mediawiki/2019/6/6a/T--Worldshaper-wuhan--R1.jpeg)

Fig.1. PCR product of part J33201 in gel electrophoresis. Lane M, Marker. Lane 1, J33201

##### 2) Identification of recombinants

As shown in Fig.2a, five positive clones were selected from the plate for bacteria colony PCR. Then two of five positive were selected for plasmid extraction, and the constructed plasmid was double digested by EcoR I/Pst I. Results showed that enzyme digestion profiles of the recombinant plasmid were in line with expectations (figure 2b).

![Results(图2)](https://static.igem.org/mediawiki/2019/4/44/T--Worldshaper-Wuhan--R2.jpeg "Results(图2)")

Fig.2. (a) Bacterial colony PCR product of recombinant plasmid in gel electrophoresis. Lane M, Marker. Lane 1-5, positive clones of recombinant plasmid. (b) plasmid enzyme digestion profile Lane M, Marker, , Lane 1-2, control plasmid double digestion(EcoR I/Pst I), Lane 3-4, recombinant plasmid double digestion(EcoR I/Pst I).

The constructed recombinant plasmid was handed over to the company for DNA sequencing. The sequencing results were aligned with BLAST software and the results showed that the constructed plasmid met our expectations.

## **2. The function of our recombinant plasmid**

##### 1) Growth curve of different concentrations of arsenite (As3+) to recombinant bacteria

Fig.3 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.3

.

![Results(图3)](https://static.igem.org/mediawiki/2019/9/91/T--Worldshaper-Wuhan--R3.jpeg)

Fig.3 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 different concentrations of arsenite (As3+) to recombinant bacteria

As shown in Fig.4, our constructed biosensor based on J33201 part is very sensitive to arsenite(As3+) and the fluoresence signal can be detected at 50ppb arsenite by instrument as well as by naked eyes(Fig.4a, 4c) . Significant dose-dependent effect was observed at the range of 50ppb-1ppm arsenite,within this range, GFP expression has a linear correlation with R2=0.9549 (Fig. b). 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.

![Results(图4)](https://static.igem.org/mediawiki/2019/9/9d/T--Worldshaper-wuhan--R4.jpeg "Results(图4)")

![Results(图5)](https://static.igem.org/mediawiki/2019/1/1a/T--Worldshaper-wuhan--R4c.jpeg "Results(图5)")

Fig.4 (a) GFP expression of E.coli in different concentration of arsenite (As3+) under the control of J33201. (b) GFP expression at arsenite concentration of 50ppb-1ppb had a liner correlation. 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.  (c)The bacteria were centrifuged, and the pellets were was observed and photographed under Blue Light Gel Imager.

#### 3) GFP expression of different concentrations of arsenite (As3+) at different time points to recombinant bacteria

As it is shown in Fig.5, 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.

![Results(图6)](https://static.igem.org/mediawiki/2019/e/e9/T--Worldshaper-wuhan--R5.jpeg)

Fig.5 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.

Bioabsorbent

As shown in figure, the photograph of SDS-PAGE and Western blot of orpF-fMT fusion protein was successfully expressed under IPTG induction with the molecular weight between 25KDa and 35kda, which was consistent with the expected molecular weight of 27.4kda, indicating that the fusion protein was successfully expressed. In addition, SpPCS protein was also successfully expressed under IPTG induction, with the molecular weight between 40 KDa and 55KDa, which was consistent with the predicted molecular weight of 46.7Da, and the protein was successfully expressed on the surface.

![Results(图7)](https://static.igem.org/mediawiki/2019/3/3e/T--Worldshaper-wuhan--R6.jpeg "Results(图7)")

Fig.6. (a)IPTG-induced protein expression of recombinant plasmid pET24a-oprF-fMT and pET42a-SpPCS in SDS-PAGE M：protein marker, 1-3 for oprF-fMT fusion protein expression, and 1: IPTG(-); 2: IPTG (1mM), 37℃,6h; 3: IPTG(1mM), 25℃,overnight; 4-6 for SpPCS protein expression, and 4: IPTG(-), 5: IPTG (1mM), 37℃,6h, 6: (1mM), 25℃,overnight. (b) IPTG-induced protein expression of recombinant plasmid pET24a-oprF-fM(b left) and pET42a-SpPCS(b right) in Western blot by using anti-Histag antibody.

As shown in figure 7, compared with the control group, the recombinant strain expressing SpPCS had a significantly higher adsorption capacity for arsenic than the control group. After adding 10μM of arsenic solution for 15 minutes, cells expressing SpPCS accumulated levels of arsenite 1.73-fold higher than those accumulated by the control. As time increased to 45min, the adsorption capacity increased to 4.08-fold compared with the control, indicating that the SpPCS produced phytochelatin(PC) to sequester arsenic functionally in the cell.

![Results(图8)](https://static.igem.org/mediawiki/2019/1/17/T--Worldshaper-wuhan--R7.jpeg)

**Fig****.****7****Arsenic accumulation in E. coli cells BL21(DE3) strain harboring the control vector(control) or expression SpPCS.** The plasmid containing pET-SpPCS was transformed into BL21(DE3) and the next day a single colony was chose to culture overnight.Then the overnight culture was inoculated into the fresh LB broth at 1:100 , OD600 was 0.6-0.8. IPTG(1mM) was added and cultured at 28℃overnight. The bacteria was centrifuged and washed with PBS and resuspended into PBS. A final concentration of 10μM arsenite solution was added into the bacteria. Samples were taken every 15min, and 1ml was centrifuged at 5000rpm for 5min. The supernatant was collected and reserved for the detection of arsenic concentration. The arsenic in the sample was determined by the atomic fluorescence spectrometry.

To summary, the protein of the biosorption device we constructed has been successfully expressed. Compared with the control group, the recombinant bacteria expressing SpPCS significantly increased the adsorption capacity of arsenic.

Taken together, in this project, we successfully established a biosensor based on the well-known arsenic response part J33201, which can quickly and sensitively detect arsenite(As3+) in water. Furthermore, the arsenic biosorption we construct to remove arsenic in water is fast and effective.

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