Team:JACOXH China/Contribution




The purpose of this project is to construct various expression elements in the beneficial bacteria Bacillus subtilis, so that the mercury sensing protein merR can activate the expression of MerB3 and AtLCD metal proteins by sensing the concentration of Hg in contaminated soil, so as to immobilize organic and inorganic Hg ions. At the same time, earthworm was used as the living bioreactor of B. subtilis to expand the population of B. subtilis, and further improve the immobilization efficiency of the components constructed in B. subtilis to contaminated soil Hg. Based on the original project (Escherichia coli as expression system, MerR as Hg sensing protein, and various amplification elements to detect Hg concentration in soil), this project has made the following improvements:
The original expression system E. coli belongs to human pathogenic bacteria, which is not suitable for release into environmental soil. Now the expression system is replaced by B. subtilis (BS), which is harmless to environment and human and animal, to express each element, so as to lay a foundation for the future application of environmental protection.
Each element in the original project was adapted to be expressed in E. coli system. Now, the expression system is adjusted to BS (Bacillus subtilis). We redesigned the elements suitable for expression in BS, and constructed the diffusion model of Hg in soil as follows:
1)From B. megaterium with high homology of BS, the Hg sensing protein merR and merB3 gene needed for organic Hg immobilization were cloned. 2)The newly added metallothionein regulatory gene AtLCD will be used to immobilize inorganic Hg ions. This gene is from Arabidopsis thaliana. We optimized its codon bias and synthesized the corresponding sequence to make it suitable for expression in BS (the original sequence is, the optimized sequence is). We used exp optimizer software to optimize the original sequence of AtLCD. The specific operations are as follows: input the original atlcd sequence (from Arabidopsis thaliana), select the host to be optimized (B. subtilis), and select the optimized parameters (including: 5 'region, DNA repeat sequence, mRNA secondary structure, GC content, SD) The optimized codon preference sequence was obtained by running the software. The optimized atlcd sequence is as follows:

3) Expression amplification element (RinA_p80α) in the original project, we replaced the RBS sequences with different intensities to adjust the expression of metallothionein regulatory gene AtLCD. According to the optimized atlcd gene sequence, 96 RBS (ribosomebinding site) sequences with different translation initiation rates were calculated. Two RBS sequences with high score and one with medium score were selected to be transformed into DNA sequence and connected with atlcd sequence to regulate the expression intensity of atlcd. The selected RBS are shown in Table 1:

4)By combining the above elements, the whole Hg sensing and fixing system is obtained as follows (Fig. 1)

Fig. 1. Construction of Hg sensing and immobilization system in pax01 plasmid

5)Construction of Hg diffusion model in soil and estimation of Hg fixation effect of BS and BS + earthworm
5.1 Construction of Hg diffusion model in soil According to Chen Manli's research (2019), brown soil (organic matter is about 16.57g/kg, clay is about 26.11%, bulk density is about 1.47g/cm3, pH > 7.5, and water content is about 23%. The experimental temperature was 20 ℃, and the pH value of the test soil was about 8.0). 10 cm at the upper end of the model was the test soil without Hg, which was divided into 10 soil layers per 1 cm; the lower 5 cm was Hg contaminated soil containing 40 mg / kg (the contaminated soil was prepared with HgCl2). The diffusion concentration of Hg in each soil layer (0.5cm soil layer in each layer for testing) and time are measured regularly. The migration amount of Hg in each soil layer is calculated according to the following formula. The results are shown in Table 2. Migration quantity (MQ): the mass of heavy metals passing through unit area in unit time, unit: g / (M2 · a). The calculation formula is as follows:

5.2 estimation of fixed amount of Hg by engineering bacteria BS in the model Assuming that BS (size set at 0.6×3µm) is evenly distributed in the test soil, if 0.5L BS bacterial solution with the concentration of 1 × 10^8 cells / L is mixed into the test soil, the concentration of BS in each soil layer is C2 5×10^7 cells / cm soil). The surface area of single BS cells (S1) =2πr2+πdh=2×3.14×0.32+3.14×0.6×3=6.22µm2 The total surface area of BS in each cm soil (S2) =S1×C2=6.22µm2×5×10^7=3.11×10^8µm2=3.11×10^-4m2 The adsorption capacity of BS to Hg in each cm soil layer (A1) = MQ×S2 According to the above formula, the adsorption capacity of BS to Hg is calculated. See Table 2 for details.

The H2S produced by AtLCD protein can react with heavy metal ions to form stable heavy metal sulfide, thus realizing the immobilization of mercury and other heavy metals. According to the literature report (Referring to "master's thesis: AtLCD and AtDCD can enhance the tolerance of Escherichia coli to Cd2+ (Shen jiejie Shanxi University, June 2013), the fixation rate of heavy metals such as Pb and CD by AtLCD is 97%, so we assume that the fixation rate of mercury ions by AtLCD is also 97%.
The amount of Hg adsorbed by Bacillus sp. (A2) = ∑ A1 × Hg migration time (T1) The fixed amount of Bacillus subtilis Hg (A3) in designated days = A2 × 0.97 Fixed amount of Hg per kg contaminated soil by Bacillus subtilis in designated days (A4) = A3 × (1000 / 115) [The weight of soil in the model is 115cm]
According to the above formula and the established model, the fixed total amount of Hg in soil by applying 0.5L 1×10^8 Bacillus subtilis was about 0.5172mg in 60 days. When converted to 1 × 10^8 Bacillus subtilis per kg soil, the fixed total amount of Hg in the soil was about 4.4974 mg/kg (Table 3).

5.3 estimation of Hg fixation in the model by engineering bacteria BS+earthworm
Other conditions are the same as those described in 5.2 model, but an earthworm is added to the model (the size is set as 0.5×5cm, and the distance of one movement is 3cm). The difference of Hg fixation in soil with and without earthworm was compared.
According to the calculation, the time required for earthworm to run a runway (calculated in vertical direction): 10cm / 3 = 3.3s; the time to run 200 runways (the whole model volume / earthworm volume) is 3.3 × 200 = 660s < 1D, so the activity time of earthworm in the model can be ignored.
According to Zeng Lingtao et al. (2016), composting earthworms with probiotics can increase probiotics (Bacillus amyloliquefaciens, Pseudomonas fluorescens) in soil by about 1.4 times.
Therefore, the interaction of earthworm and BS can increase the population of Bacillus subtilis by 1.4 times. In other words, the fixation capacity of Hg in soil was 1.4 times higher than that of single application of BS.
That is to say, one earthworm + bacillus (1 × 10^8 cells / L) could be fixed 4.4974 * 1.4 = 6.2964mg Hg per kg soil in 60 days.

Reference: 1.Brown N L,Stoyanov J V, Kidd S P, Hobman J L. The MerR family of transcriptional regulators. FEMS Microbiology Reviews 27 (2003) 145-163. 2.Haytham M W, Lauriane L, Michael S, Ahmed M, Laurent C, Julien L V, Kevin J W, Jurgen S, Dean E W, James G O. Biochemistry. 2016 Feb 23;55(7):1070-81. 3.Zeng Lingtao, Wang Dongsheng, Wang Zhenyi, Wang Siqi, Sheng xiongjie, Chang Jiangjie, Li Huixin, Hu Feng, Jiao Jiaguo. Effects of earthworm compost combined with probiotics on soil fertility and microbial characteristics. Soil. 2016, 48 (6): 1100 – 1107 4.Chen Manli. Migration characteristics of heavy metals in topsoil. Master Thesis of Zhengzhou University, 2019 5.Shen jiejie. Atlcd and atdcd can enhance the tolerance of Escherichia coli to Cd2 +. Master's thesis of Shanxi University, 2013



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