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To reduce the amount of trimethylamine (TMA) in the gut, we considered the degradation of TMA produced by gut microorganisms. So, E. coli containing trimethylamine dehydrogenase gene could catalyse the oxidative demethylation of TMA to dimethylamine and formaldehyde.

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The trimethylamine dehydrogenase by 2022 HZAU-iGEM team

Plasmid(4μg) was synthesized by Genscript. Firstly, we centrifuged tmd plasmid powder at 5000rpm for 1 min, then added 40μg sterile ddH2O to dissolve it. The plasmid concentration was 100ng/μL. After diluting plasmid solution into10ng/μL, we transformed plasmids into competent cells E. coli BL21. The outcomes of colony PCR is showed below.

Figure 1. Colony PCR of E. coli BL21containing tmd plasmids.

We performed SDS-PAGE to identify that trimethylamine dehydrogenase can be expressed. Because trimethylamine dehydrogenase (TMADHexist as dimers, the protein molecular weight would double. So, protein molecular weight of TMADH is 164.9kDa.

Figure 2. Control is E. coli BL21 without tmd. tmd is induced E. coli BL21 with tmd.

We cultivated E. coli BL21 containing tmd, V344C tmd and E. coli BL21 without tmd (Blank) for about 3 hours (OD600 0.6~0.8). Then they were induced by 4mM theophylline for 9 hours. After adjusting the density of three tubes of bacteria and making them almost have no difference, we added some TMA into bacteria cultures to make the concentration of substrate TMA 5×10-5mol/L and continued to cultivate them. Take samples before we add TMA, and add TMA for 0 min, 10 min, 20min, 3h, 6h, 9h.

This is how we handle bacteria samples. 700 µl bacteria samples were centrifugated at 3000 × g 5 min at 4 °C, take 500µl supernatant. Then 300 µl freshly prepared 10 mM solution of FMOC-Cl in acetonitrile was added, after 1 min, 100 µl 100 mM glycine solution was added to neutralize the reaction.

This is our method of HPLC Supernatant was transferred to new tube for analysis on HPLC system. 10 µl was loaded on to C18 column equilibrated with acetonitrile-buffer (50%) at flow rate 0.75 ml/min. The column was then flushed with a gradient to 100% elutant buffer B (acetonitrile 75% v/v) within 5 min. Ultraviolet absorption of column elutant was monitored (220 nm) and DMA quantification was calculated based on ratio to standard sample peak area. Here comes our standard sample peak area.

Figure 3. This is the HPLC standard curve peak obtained by our team. In the literature, the retention time of DMA is about 6 minutes. However, due to the error of the culture medium, here we selected the peak change of retention time around 7 as the standard curve.

According to the above peaks, we have drawn the curve of concentration and peak area of standard solution.

Figure 4. The standard DMA LB meduim curve.

According to the standard curve, we detected the enzyme activities of wild type tmd and mutant V344C respectively. The following are our experimental results.

Figure 5. Change of DMA concentration in solution of wild type tmd and mutant after 9 hours.

According to the picture analysis, since the concentration of DMA is detected by HPLC, the concentration of DMA under the action of trimethylamine dehydrogenase should gradually increase with time. As shown in the figure, the concentration of dimethylamine in both wild type TMADH and mutated TMADH increased gradually in 0 to 6 hours. This result fully shows that our trimethylamine can effectively express and play a role in degrading TMA. Among them, the mutant V344C is also more efficient than the wild type, which shows that our numerical simulation results are also correct.

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