Team:Mingdao/Proof

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• About Us
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The AOX gene was successfully synthesized by IDT and cloned onto pSB1C3 with a fusion protein of Lpp-OmpA (LO) which is driven by a constitutive promoter and RBS. In the following experiments to prove the concept, we tested the gene expression system by a reporter gene (GFP) and analyzed AOX gene expression by SDS-PAGE and Coomassie Blue staining. Furthermore, we examined AOX enzyme activity by H₂O₂ assay, a product generated in the process of the oxidation of alcohol catalyzed by AOX. Finally, we designed a prototype to demonstrate the application of AOX enzyme on the alcohol meter in an electrochemical simulator.

The AOX gene were cloned onto pSB1C3 driven by a constitutive promoter (BBa_J23101) and RBS (BBa_B0034) and fused with bacterial outer membrane proteins, Lpp-OmpA (LO) to display protein on the cell surface of E. coli. To test whether the gene expression system works, we cloned a reporter gene (GFP) in the same context (i.e., Pcons-RBS-LO-GFP/pSB1C3). And the gene expression level was compared to Pcons-RBS-GFP/pSB1C3 (BBa_K1694035) got from NCTU-Formosa, which has the same promoter and RBS but without LO. The clones of E. coli DH5α were cultured in LB media supplemented with 34 μg/ml chloramphenicol at 37°C overnight. Because overnight cultured LB medium has a background level of fluorescence, the bacterial GFP was measured in the PBS buffer (Enzyme Microb Technol. 2001). As Figure 1 showed, the GFP expression was extremely high in E. coli. LO-GFP fusion protein expression was observed at low but significant level compared to wild-type E. coli or E. coli expressing LO outer membrane proteins.

Figure 1: Gene expression analysis. GFP gene expression was read at Ex/Em = 488/528 nm in BioTek Microplate Spectrophotometer. Lane 1: wild-type E. coli as a mock control; Lane 2: GFP expression in E. coli [BBa_K1694035]; Lane 3: LO outer membrane protein expression [BBa_K1991007] in E. coli; Lane 4: LO-GFP fusion protein expression [BBa_K1991010] in E. coli.

To analyze the AOX gene expression, we run on a SDS-PAGE gel and observed by Coomassie Blue staining. The overnight-cultured E. coli were centrifuged and lysed with Lysis Buffer (12.5 mM Tris pH 6.8, 4% SDS). The resulting lysates were subjected to SDS-PAGE with a 10% polyacrylamide gel. The gel was stained with 0.25% Coomassie Brilliant Blue R250 for 2 hours and destained until the protein bands were clear. As the data showed in Figure 2, LO protein was expressed at around the estimated molecular weight of 17 kDa, LO-AOX fusion protein at 91 kDa and AOX protein at 79 kDa. The protein expression level was consistent with the data of GFP in Figure 1, demonstrating the low gene expression level of a LO fusion protein. However, so far we cannot confirm whether LO fusion protein is able to direct AOX or GFP proteins displayed on the cell surface of E. coli. We’re planning to do a subcellular fractionation to separate the outer membrane proteins for analysis in the future.

Figure 2: AOX protein analysis. SDS-PAG and Coomassie Blue staining were used to observe protein expression level. Lane 1: wild-type E. coli as a mock control; Lane 2: LO outer membrane protein [BBa_K1991007] (17 kDa) expression in E. coli; Lane 3: LO-AOX fusion protein [BBa_K1991009] (91 kDa) expression in E. coli.; Lane 4: AOX protein [BBa_K1991003] (79 kDa) expression in E. coli.

After AOX gene expression was confirmed, we want to know its enzyme activity. AOX catalyzes the oxidation of alcohol in the following chemical reaction C₂H₅OH + O₂ → CH₃CHO + H₂O₂ and generates hydrogen peroxide. We’d like to measure the production of hydrogen peroxide to demonstrate AOX enzyme activity. Fluorimetric Hydrogen Peroxide Assay Kit of Sigma-Aldrich was used for the study. In the kit, the red peroxidase substrate is designed to react with hydrogen peroxide to generate red fluorescence signal that can be detected at Ex/Em = 540/590 nm in the microplate reader. First, a serial dilution of hydrogen peroxide was tested to see if the kit works in our lab. As Figure 3 showed, the three-fold serial dilutions of hydrogen peroxide from 10 to 0.014 μM were linearly correlated to red fluorescence intensity, demonstrating H₂O₂ concentration can be measured using this kit in our lab.

Figure 3: H₂O₂ assay. The concentrations (μM) of hydrogen peroxide were measured at red fluorescence intensity unit (RFU) [Ex/Em = 540/590 nm]. The serial dilutions of hydrogen peroxide were tested and correlated to red fluorescence intensity.

 

 

Next, we examined the activity of AOX enzyme by measuring H₂O₂ production from the oxidation of ethanol. We prepared 1ml of overnight cultured E. coli displaying LO-AOX enzyme. The bacteria were centrifuged and resolved in Assay Buffer provided by the Sigma-Aldrich kit. Then, the resulting lysates were mixed with the increasing concentrations of ethanol from 0%, 0.025%, 0.05%, 0.075%, 0.10%, 0.15% to 0.2% for 3 minutes at room temperature. The tested alcohol concentrations were at the range from 0.03% (the alcohol law limit for safety driving) to 0.2% (people may lose consciousness at this level). The following procedure was according to the manufacture’s instruction. The data were read out at Ex/Em = 540/590 nm in the microplate reader (BioTek Microplate Spectrophotometer). As Figure 4 showed, the intensities of AOX activity were significantly and linearly correlated to the concentration of ethanol in a dose-dependent manner. The results indicated that LO-AOX enzyme we produced has a functional enzyme activity and reacted with alcohol concentration dose-dependently, implying we can use this assay to measure the unknown sample of alcohol. Although we cannot make sure whether AOX enzyme was displayed on the cell surface of E. coli or not in this moment. In addition, it is noted that according to the manufacture’s instruction, the substrate (ethanol), bacteria (E. coli) or LO-expressed E. coli has a basal level of fluorescence intensity. To count the measures clearly, you should subtract the background intensity.

Figure 4: AOX enzyme activity assay. The various concentrations of ethanol were reacted with E. coli displaying AOX enzymes. The assay was performed using Fluorimetric Hydrogen Peroxide Assay Kit (Sigma-Aldrich). The readout was presented in arbitrary unit to show the enhanced AOX activity caused by alcohol concentration in a dose-dependent manner.

 

 

Taken together, we cloned the AOX genes, tested our expression system and performed protein analysis followed by the enzyme activity assay. Now, we’re ready to examine the chemical properties of AOX enzyme in an electrochemical experiments and apply it to the analyzer.