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United_Shanghai_HS

Contribution

The purpose of this project is to use the beneficial bacterium Bcillus subtilis as a vector to construct various expression elements in its body. When transferred into the transformed plasmid, the Bacillus subtilis can express downstream in the natural environment due to the loose regulation of the PsacB promoter. The two enzymes PETase and MHETase initially degraded some ethylene glycol. As a certain amount of ethylene glycol is degraded, the ethylene glycol hid the terminator contained in the riboswitch and further activate the glycine riboswitch. Express more PETase and MHETase to achieve the regulation of protein expression, that is: without PET, the two enzymes are expressed in small amounts, and with PET, the expression of enzymes is determined by the amount of environmental PET. As a reporter gene, mcherry can indicate the need to add sucrose to amplify the effect, and more ethylene glycol increased the transcription of PETase and MHETase. Present a positive feedback effect. At the same time, earthworms are used as the living bioreactor of B. subtilis to expand the population of B. subtilis and further improve the fixing efficiency of each element constructed in its body to the contaminated soil PET. This project has made the following improvements on the basis of the original project (using Escherichia coli as the expression system to express PETase and MHETase to degrade PET):

The original expression system E. coli belongs to human pathogens and is not suitable for release into environmental soil. Now the expression system is replaced with B. subtilis (Bs) which is harmless to the environment and humans and animals to express each element, which is environmentally friendly for future applications. Lay the foundation.
All elements in the original project are adapted to be expressed in the E. coli system. Now the expression system is adjusted to Bs (Bacillus subtilis). We have redesigned the elements suitable for expression in Bs, and constructed the engineering bacteria to infiltrate the soil. Model of degradation of plastics.

We found in the literature that the Wu Bian team of the Institute of Microbiology of the Chinese Academy of Sciences optimized the enzymes required by the Osaka bacteria to degrade PET plastic, and obtained a redesigned enzyme with significantly enhanced stability, which made the Osaka bacteria's effect on the PET film. The degradation efficiency has been increased by 300 times. We used its optimized ISF6_4831 protein sequence; found the protein sequence of Osakaji ISF6_0224; GFP, the protein sequence of mCherry fluorescent protein.
We fused the gene sequences of ISF6_4831, GFP; ISF6_0224, mCherry fluorescent protein respectively. In the literature, we found that the signal peptide SPLamB has the highest exocrine efficiency. We deleted the signal peptide sequence before the ISF6_4831 (expressing PETase) and ISF6_0224 (expressing MHETase) gene fragments, and added the signal peptide SPLamB gene before the fusion sequence Sequence to improve the exocrine expression of the protein. The optimized in-frame gene sequence is as follows:

①　Component name: ISF6_4831 (after optimization)
Short description: In-frame expression gene of PETase, 843 bp
Specific description: ISF6_4831 has a high sequence similarity with the angle with PET hydrolysis activity, but it has a high sequence similarity to the water of PET. The deactivation activity and selectivity are significantly higher than other PET hydrolases.

ATGCAAGCTGCTGTTCTTGGCGGCCTTATGGCTGTTTCTGCTGCTGCTAC 50
AGCTCAAACAAACCCTTACGCTCGTGGCCCTAACCCTACAGCTGCTTCTC 100
TTGAAGCTTCTGCTGGCCCTTTCACAGTTCGTTCTTTCACAGTTTCTCGT 150
CCTTCTGGCTACGGCGCTGGCACAGTTTACTACCCTACAAACGCTGGCGG 200
CACAGTTGGCGCTATCGCTATCGTTCCTGGCTACACAGCTCGTCAATCTT 250
CTATCAAATGGTGGGGCCCTCGTCTTGCTTCTCATGGCTTCGTTGTTATC 300
ACAATCGATACAAACTCTACATTCGATTACCCTTCTTCTCGTTCTTCTCA 350
ACAAATGGCTGCTCTTCGTCAAGTTGCTTCTCTTAACGGCGATTCTTCTT 400
CTCCTATCTACGGCAAAGTTGATACAGCTCGTATGGGCGTTATGGGCCAT 450
TCTATGGGCGGCGGCGCTTCTCTTCGTTCTGCTGCTAACAACCCTTCTCT 500
TAAAGCTGCTATCCCTCAAGCTCCTTGGGATTCTCAAACAAACTTCTCTT 550
CTGTTACAGTTCCTACACTTATCTTCGCTTGCGAAAACGATTCTATCGCT 600
CCTGTTAACTCTCATGCTCTTCCTATCTACGATTCTATGTCTCGTAACGC 650
TAAACAATTCCTTGAAATCAACGGCGGCTCTCATTCTTGCGCTAACTCTG 700
GCAACTCTAACCAAGCTCTTATCGGCAAAAAAGGCGTTGCTTGGATGAAA 750
CGTTTCATGGATAACGATACACGTTACTCTACATTCGCTTGCGAAAACCC 800
TAACTCTACAGCTGTTTCTGATTTCCGTACAGCTAACTGCTCT

②　Component name: ISF6_0224 (after optimization)
Short description: MHETase expressed gene in frame, 1788bp
Specific description: MHETsae was also found in this bacteria, and it was confirmed that it can be coupled with PETase to express and play a role in further decomposition. MHETase can further decompose mhetase, the main product metabolized by PETase, and degrade it into TPA and EG. TPA and EG can enter the bacteria's own metabolic pathway for complete degradation.

ATGCTTCTTGCTTCTGTTGCTCTTGCTGCTTGCGCTGGCGGCGGCTCTAC 50
ACCTCTTCCTCTTCCTCAACAACAACCTCCTCAACAAGAACCTCCTCCTC 100
CTCCTGTTCCTCTTGCTTCTCGTGCTGCTTGCGAAGCTCTTAAAGATGGC 150
AACGGCGATATGGTTTGGCCTAACGCTGCTACAGTTGTTGAAGTTGCTGC 200
TTGGCGTGATGCTGCTCCTGCTACAGCTTCTGCTGCTGCTCTTCCTGAAC 250
ATTGCGAAGTTTCTGGCGCTATCGCTAAACGTACAGGCATCGATGGCTAC 300
CCTTACGAAATCAAATTCCGTCTTCGTATGCCTGCTGAATGGAACGGCCG 350
TTTCTTCATGGAAGGCGGCTCTGGCACAAACGGCTCTCTTTCTGCTGCTA 400
CAGGCTCTATCGGCGGCGGCCAAATCGCTTCTGCTCTTTCTCGTAACTTC 450
GCTACAATCGCTACAGATGGCGGCCATGATAACGCTGTTAACGATAACCC 500
TGATGCTCTTGGCACAGTTGCTTTCGGCCTTGATCCTCAAGCTCGTCTTG 550
ATATGGGCTACAACTCTTACGATCAAGTTACACAAGCTGGCAAAGCTGCT 600
GTTGCTCGTTTCTACGGCCGTGCTGCTGATAAATCTTACTTCATCGGCTG 650
CTCTGAAGGCGGCCGTGAAGGCATGATGCTTTCTCAACGTTTCCCTTCTC 700
ATTACGATGGCATCGTTGCTGGCGCTCCTGGCTACCAACTTCCTAAAGCT 750
GGCATCTCTGGCGCTTGGACAACACAATCTCTTGCTCCTGCTGCTGTTGG 800
CCTTGATGCTCAAGGCGTTCCTCTTATCAACAAATCTTTCTCTGATGCTG 850
ATCTTCATCTTCTTTCTCAAGCTATCCTTGGCACATGCGATGCTCTTGAT 900
GGCCTTGCTGATGGCATCGTTGATAACTACCGTGCTTGCCAAGCTGCTTT 950
CGATCCTGCTACAGCTGCTAACCCTGCTAACGGCCAAGCTCTTCAATGCG 1000
TTGGCGCTAAAACAGCTGATTGCCTTTCTCCTGTTCAAGTTACAGCTATC 1050
AAACGTGCTATGGCTGGCCCTGTTAACTCTGCTGGCACACCTCTTTACAA 1100
CCGTTGGGCTTGGGATGCTGGCATGTCTGGCCTTTCTGGCACAACATACA 1150
ACCAAGGCTGGCGTTCTTGGTGGCTTGGCTCTTTCAACTCTTCTGCTAAC 1200
AACGCTCAACGTGTTTCTGGCTTCTCTGCTCGTTCTTGGCTTGTTGATTT 1250
CGCTACACCTCCTGAACCTATGCCTATGACACAAGTTGCTGCTCGTATGA 1300
TGAAATTCGATTTCGATATCGATCCTCTTAAAATCTGGGCTACATCTGGC 1350
CAATTCACACAATCTTCTATGGATTGGCATGGCGCTACATCTACAGATCT 1400
TGCTGCTTTCCGTGATCGTGGCGGCAAAATGATCCTTTACCATGGCATGT 1450
CTGATGCTGCTTTCTCTGCTCTTGATACAGCTGATTACTACGAACGTCTT 1500
GGCGCTGCTATGCCTGGCGCTGCTGGCTTCGCTCGTCTTTTCCTTGTTCC 1550
TGGCATGAACCATTGCTCTGGCGGCCCTGGCACAGATCGTTTCGATATGC 1600
TTACACCTCTTGTTGCTTGGGTTGAACGTGGCGAAGCTCCTGATCAAATC 1650
TCTGCTTGGTCTGGCACACCTGGCTACTTCGGCGTTGCTGCTCGTACACG 1700
TCCTCTTTGCCCTTACCCTCAAATCGCTCGTTACAAAGGCTCTGGCGATA 1750
TCAACACAGAAGCTAACTTCGCTTGCGCTGCTCCTCCT

By combining the above components, the overall PET degradation system is obtained as follows (Figure 1):
Figure 1. PET degradation system constructed in pWB980 plasmid
Establish a mathematical model
The content and object of the research is the model of engineering bacteria infiltrating the soil to degrade plastics, and the expanded model is the degradation model of the soil under the presence of earthworms. In the soil, the process of colony transfer and degradation is mainly composed of four components, namely convection, diffusion and penetration, and degradation. The test soil is brown soil (organic matter is about 16.57g/kg, clay accounts for about 26.11%), bulk density is about 1.47g/cm3, p H>7.5, and water content is about 23%. The test temperature is 20°C, and the pH of the test soil is about 8.16.

The description of the infiltration and degradation process of bacterial colonies in the soil is mainly done by looking for the mechanism in a large number of statistical models.
The general structure is:
①　Convection process:

In equation (1), the first one on the left represents the convection process in the infiltration process. In relatively sandy soil, the first batch of colonies moves into the soil by convection. In this process, the colonies move through gravity , Groundwater and air flow through the gaps for convection transfer, and the driving force is described by the gradient of convection. The relationship between the calculated velocity vector v is:

φ is the osmotic pressure in the convection process:

②Diffusion process

As the convective motion spreads out, this process is diffusion. It is described by the second term on the right side of equation (1). The infiltration process of the colony is assumed to be the sum of two factors, which are the expansion tensors of convection on both sides. , Can be expressed as:

In the process of colony flow in the soil, the movement diffusion is much greater than the molecular diffusion. The D term is whether there are earthworms.

③　Degradation process

The degradation process is the most complicated in the whole process. Degradation is the change and fusion of different phases. The fusion of phases is carried out according to the balance of the system. The process of degradation is the gradual change of the concentration of the degraded material in the soil. The process is:

S is the aggregate amount of degraded substances, and C is the concentration component in the soil. K is the distribution process of degradation. If the soil is highly polluted, the definition of soil isotherm needs to be considered.

For the hypothesis proposed in this article, the rate of pollutant degradation can be expressed in differential form, as follows:

The differential form of the first-order dynamic model of the model is:

The above dynamic model is used to describe the degradation process, bringing in the previous expansion modulus energy type, and the final diffusion model is:

The following specific examples are used to reproduce the above mathematical model and perform calculations.

一、Individual soil modeling results and analysis

Table 1 Analysis results obtained by modeling

2.Adding the modeling results and analysis of earthworms

Figure 2: Earthworm penetration model Infiltration(I) and Flux(q)

In the figure above, the horizontal axis is the time of penetration, the dashed line on the vertical axis is the amount of earthworms that have already penetrated, and the solid line on the vertical axis is the penetration rate.

Table 2 Analysis results obtained by modeling (including earthworms)

Compare Results

Figure 3: Comparison of penetration rate of Bacillus subtilis in the absence and presence of earthworms

In the above figure, the red line is the penetration rate of fungi in normal soil. As time goes on, the penetration depth of the monitored soil colony is gradually increasing, but the penetration rate in the soil will gradually be affected by the resistance or isolation of the soil. Decrease, when the depth increases to a certain extent, the rate of decrease will become smaller and smaller, tending to stabilize.

In the same way, the blue line is the penetration rate of the presence of earthworms. In the presence of earthworms, the penetration rate will gradually decrease, but the overall penetration rate is greater than that of pure soil.

Figure 4: The relationship between the penetration rate of bacterial species and the proportion of degraded pollution and the proportion of effective degrading bacteria

The above figure gives an explanation of the degradation of the colony. The horizontal axis is the permeability of the colony. The permeability is gradually increasing. The upper part shows the specific gravity of the degraded pollution occupying the entire 1 square meter measurement. The following figure shows The effective degrading colony occupies the proportion of the total colony. The blue color contains earthworms, the black color does not contain earthworms, and the red color contains half the number of earthworms as blue.

Team:United Shanghai HS/Contribution