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Results

Our results are divided into four parts: Parts, Characterization,

Growth curves, and Device.

Parts

1. BioBricks

Here are the standard BioBrick Parts we have created and submitted this year.

	Name	Type	Description	Length
1	BBa_K1393000	Coding	oprF(Val188)+GS linker+CBP	600
2	BBa_K1393001	Coding	oprF(Ala196)+CBP	609
3	BBa_K1393002	Composite	cyn operon in E. coli BL21(DE3) (cynR+cynT+cynS)	2169
4	BBa_K1393003	Coding	Outer Membrane porin Protein C from E. coli BL21(DE3)	936

Besides, we are standardizing another four new standard BioBrick Parts: FLA, PpcoA, oprF-CBP-HA and oprF-GS-CBP-HA.

Characterization of parts

2. 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.

Preliminary Test 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 and 0.1mM. 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.
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 2.1: Promoter test of PpcoA promoting induced by Cu²⁺. We can find that the promoting ability of PpcoA arises according to the concentration raise of Cu²⁺. This preliminary data was performed under the help from HZUA-China team.

Experiment 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 2.2: Promoter test of PpcoA promoting induced by Cu²⁺. In our teams experiment, we optimized the experiment procedure based on preliminary test and we can find that the relative fluorescent intensity increases obviously with the increase of the concentration of copper ions, which indicates that 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.

3. Characterization of flA and oprF

3.1.1 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.

Figure 3.1: SDS page test of purified recombinant oprF and flA protein.

4. 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 fluorescent microscope.

Figure 4.1: model and result of immunofluorescence analysis of oprF displayed on E. coli

5. Cu²⁺ adsorption capacity assay

We have got the evidence of SDS-PAGE and immunofluorescence assay, which 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. Then, we further investigated its capacity to absorb 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).

Figure 5.1: demonstration of BCO reaction test

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.

Figure5.2 standard curve of certain Cu²⁺ concentration shows the stability of BCO test. While the time-absorbance curve of BCO test of pET-28a-oprF-GS(linker)-CBP culture shows the successful immobilization of Cu²⁺

Growth curves

6. Growth curves test

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 E. coli BL21 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.

6.1 Growth curves test of flA

6.1.1 The growth curve of fluorine-containing sewage gradient of transformants

Figure 6.1: The growth curve of flA expression strains and control in LB culture medium, compared to control, flA expression did not significantly reduce the growth of our bacteria.

Figure 6.2: The results were right on target with our expectations. Presence of fluoride ion pollutant environment, flA transformants did have a better growth advantage than control.

6.2 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 6.3: Growth curve of normal bacteria carrying vector in culture environment with or without Cu2+.

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

Figure 6.4: Growth curve of bacteria carrying vector/oprF-CBP/oprF-GS-CBP in culture environment with different Cu2+ concentration.

Contrary to our expectation, the growth of bacteria carrying oprF-CBP grew as normal as control bacteria carrying pET28a vector in Cu²⁺ environment. 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. To cope with the problem, we added a GS-linker between oprF and CBP to reduce the spatial interaction. 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 oprF-CBP or pET28a vector. 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⁺.

Device

7. Rotating Biological Contactor (RBC)

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.