Team:HUST-China/Result

Results
Our results are divided into three parts: Characterization of parts, Growth curves, and Device.
1. Characterization of parts
1.1 Characterization of the copper sensitive promoter PpcoA
To determine that whether promoter PpcoA can respond to different Cu²⁺ concentration, we constructed the recombinant plasmid pET28a-PpcoA-mRFP and read the fluorescent intensity for assaying the promoter's transcription ability when incubated in the LB medium containing Cu²⁺. Team HZAU-China helped to sequence the plasmid and do some pre-experiment to characterize the promoter.

The following is the work of HZAU.
Procedure
1. Add 2mL LB culture medium, 2uL Kanamycin (50mg/L) and 20uL bacterial samples to a 5mL centrifuge tube. Cultivate in the 37°C shaking incubator and set the rotational speed at 180 rpm/min.
2. Add CuSO₄ solution to induce in concentration of 0, 0.02, 0.1 and 1mM. Set three copies for each concentration as repetitions. Cultivate in the 37°C shaking incubator at the rotational speed of 180 rpm/min for 4 hours.
3. Add 200uL bacterial samples from each centrifuge tube to the 96-well plate. Set two copies for each centrifuge tub as repetitions. Meanwhile, add LB culture medium containing and not containing 1.0mM CuSO₄ as blank controls.
4. Read the OD₆₀₀ and fluorescent intensity (with the emission wavelength at 607nM and excitation wavelength at 584nM) using a multifunctional microplate reader.
5. Divided the fluorescent intensity result using the value of OD₆₀₀. Record the data for compare and analysis.

Result and discussion

Figure 1,2: As the blank control sample handles all groups with fluorescence value =104.5, OD₆₀₀=0.072. From the data can be seen as the copper concentration increased, the fluorescence value have a rising trend, we slightly changed the protocol, and then expand the concentration gradient to get data.

Procedure
1. Cultivate bacterial samples overnight in the 37°C and set the rotational speed at 200 rpm/min.
2. Add 5mL LB culture medium, 5uL Kanamycin (50mg/L) and 100uL bacterial liquid from step 1 to a 20mL culture flask. Cultivate in the 37°C shaking incubator at the rotational speed of 200 rpm/min. When the OD₆₀₀ is 0.5 to 0.8, add copper ions in different concentration. Set two copies for each concentration as repetitions. Cultivate in the 37°C shaking incubator at the rotational speed of 200 rpm/min for 3~4 hours.
3. Add 100uL bacterial samples from the culture flask to a 96-well plate. Set three copies for each culture flask as repetitions. Meanwhile, add LB culture medium containing and not containing CuSO₄ as blank controls.
4. Read the OD₆₀₀ and fluorescent intensity (with the emission wavelength at 607nM and excitation wavelength at 584nM) using a multifunctional microplate reader.
5. Divided the fluorescent intensity result using the value of OD₆₀₀. Record the data for compare and analysis.

Result and discussion

Figure 3: As the picture showed above, the relative fluorescent intensity increases with the increase of the concentration of copper ions, which means the transcriptional rate is positively correlated to the concentration of copper ions.
The experimental result was in line with our expectations, so that we can ensure the circuit working normally.

1.2 Characterization of FLA and oprF
1.2.1 Protein expression of oprF
Plasmid pET28a-oprF was transformed into E. coli BL21(DE3) for protein expression analysis. The strain was grown in Luria broth containing 100ug/ml kanamycin at 37℃, 250rpm/min until an absorbance of 0.4–0.6 at 600 nm was reached. We then added IPTG to 0.5mM and continued the incubation at 28℃ overnight to induce the overexpression of oprF. The cells were collected, suspended with 10mM imidazole containing 0.1mM protease inhibitor PMSF and then disrupted using Selecta Sonopuls. After centrifugation, the sediment was treated with 1*SDS gel loading buffer and kept in boiling water for 5 minutes and applied to SDS-PAGE.

1. Immunofluorescence analysis of oprF
To identify that whether our oprF has anchored on the cell membrane of E. coli, we performed immunofluorescence assay. HA tag was added to the N-terminal of oprF-GS(linker)-CBP so that the recombinant protein oprF-GS(linker)-CBP-HA can be specifically recognized by anti-HA antibody. When FITC labeled anti-IgG antibody was used as the secondary antibody and interacted with the primary antibody, green fluorescence could be observed in the cell membrane of E. coli under the fluorescent microscope.

Control

2. Cu²⁺ adsorption capacity assay
Through the evidence of SDS-PAGE and immunofluorescence assay, we confirmed that the recombinant protein oprF-GS(linker)-CBP can be expressed in high level and successfully anchored on the cell membrane in our engineered bacteria. Thus, we further investigated its capacity to adsorb Cu²⁺ when incubated in Cu²⁺-rich environment. Samples of culture medium in different stages was collected and assayed to identify the Cu²⁺ concentration using BCO method (see more detail of BCO method).

Time-Absorbance curve:
1

2

3. (Initial concentration: 3ug/ml)

Attached: BCO method
Principle: The complexation between Oxalic acid bis-(cyclohexylidene hydrazide) (BCO) and Cu²⁺ will be triggered when the pH reaches to 7~9 and generates a kind of blue clathrate, of which the absorbance at 600nm is linearly correlated with the concentration of Cu²⁺. It’s a generally used method for the determination of trace amount of Cu²⁺ in polluted water. We chose pH 8.8 for reaction.

Procedure:
1. E.coli transformed with pET-28a-oprF-GS(linker)-CBP was grown on LB medium ( 100mg/ml kanamycin, 250r/min, 37℃ );
2. When the absorbance at 600nm reaches to 0.6, add IPTG to 0.5mM and continue the incubation overnight ( 28℃, 200r/min );
3. Added Cu²⁺ to 5ug/ml , draw 1ml culture medium and collect the supernatant as sample "0" for subsequent assay.
4. Collect more samples in every 30 minutes;
5. Add 200ul tris-HCl (pH 8.8 ) and 50ul 0.2% Oxalic acid bis-(cyclohexylidene hydrazide) (BCO) to every samples, incubate it at 55℃ for 30 minutes;
6. Measure the absorbance at 600nm using spectrophotometer.

2. Growth curves
2.1 Growth curves of FLA
To ensure the survival of our E. worker in industrial sewage, we grew E. coli carrying parts in medium containing corresponding pollutant, and measured the OD₆₀₀ of the culture to draw the growth curve. We cloned the part responsible for the removal of each kind of pollutant, specifically copper, cyanide and fluoride, into vector pET28a respectively. The part is under the regulation of T7 promoter. After transformed into BL21 E. coli strain, IPTG was added to induce expression. E. coli carrying pET28a backbone was used as negative control.
The number of bacteria in medium is judged by OD₆₀₀. The higher the OD₆₀₀, the more bacteria the medium contains. So we take the OD₆₀₀ value as the bacteria growth.

2.1.1 The growth curve of fluorine-containing waste water gradient of transformants

Figure 1: The growth curve of FLA transformants and pET28a transformants in LB culture medium. FLA expression strains in normal water, compared with pET28a, did not significantly reduce the growth advantage.

Figure 2, 3: Fluoride in wastewater is indeed harmful to cell (FLA and pET28a) growth.

2.1.2 The growth curve of fluorine-containing waste water gradient of FLA and pET28a transformants

Figure 4: The results were right on target with our expectations. Presence of fluoride ion pollutant environment, FLA transformants did have a better growth advantage than pET28a transformants.

2.2 Growth curves of oprF-CBP and oprF-GS(linker)-CBP
We constructed the plasmid pET28a-oprF-CBP and get the growth curve of it first.

Figure 1: Under high concentration of copper ions, bacteria significantly grow worse than that in normal water. It revealed that the high concentration of copper ion is indeed harmful to the growth of bacteria.

Figure 2: Contrary to our expectation, the growth of E. coli carrying oprF-CBP is consistently worse than that of E. coli carrying pET28a backbone. The possible reason is that fusion-expressed CBP and oprF are so close to affect the function of CBP. Without CBP capable of binding to copper ions, the E. coli carrying oprF-CBP loses its tolerance of high concentration of Cu²⁺. Meanwhile, the expression of heterologous protein reduces the competitive advantage of our transformant. These two mechanisms work together to result in the slow growth of E. coli carrying oprF-CBP.

Figure 3: To cope with the problem, we added a GS-linker between oprF and CBP to reduce the spatial interaction; and we used the same protocol to draw a new growth curve. We were very excited to see that our method works! The growth of E. coli carrying oprF-GS-CBP is significantly and consistently better than that of E. coli carrying pET28a backbone. It proves that 1) a GS-linker is necessary for the normal function of CBP when expressed as a fusion protein with oprF; 2) our oprF-GS-CBP motif can effectively increase E. coli's the tolerance of Cu²⁺.

3. Device
Rotating Biological Contactor (RBC)
Background
The removal of heavy metals from diluted industrial sewage is impractical due to the inadequacy of traditional treatments (e.g. chemical precipitation and evaporation). Luckily, we have Bio-adsorption now, an alternative method that is highly efficient and produces no secondary pollution. The rotating biological contactor (RBC), which is very potent, efficient and can make good use of byproducts like sludge, is now widely used in sewage treatment. In RBC, toxic metals are sequestered and adsorbed onto biological materials, which are cheap and easy to deal with. In this way, the adsorbed metals can be recycled and the biological materials can be reused. However, traditional RBC can only be applied to domestic sewage. To expand RBC's application to industry, in our project, we redesigned and improved the traditional RBC.
Here are the 3D modelling of our RBC:

Entity photo

Consult the Toolkit for more details.