UreE-UreF-UreG-UreD

Usage

UreEFGD serves as the accessory factor subunit for bacterial urease proteins. When UreABC(BBa_K5166066), which forms the catalytic heterotrimer, is co-expressed with the accessory subunit UreEFGD, it results in the production of functional bacterial urease. In this project, we utilized E.coli to express this urease enzyme, allowing for the selective precipitation of metal ions in the form of carbonates, thereby achieving the goal of mineralization.

Biology

UreE, UreF, UreG,UreD are all derived from S. pasteurii, and their sequences can be found in the following parts:(UreE:BBa_K5166018;UreF:BBa_K5166019;UreG:BBa_K5166020;UreD:BBa_K5166021).Microorganisms that produce urease can utilize the enzymatic activity of urease to catalyze the decomposition of urea into ammonia and carbonate, causing metal ions to precipitate into stable carbonates, effectively immobilizing these metals^[1].It has been reported that the high-urease-activity strain S. pasteurii can utilize this hydrolytic capability to bind heavy metal ions in water and generate carbonate precipitates to remove heavy metals^[2]. Based on the main part of UreABC,UreD, UreF, and UreG form a complex that functions as a GTP hydrolysis-dependent molecular chaperone. This complex activates the urease apoprotein by assisting in the assembly of the nickel-containing metal center within UreC. The UreE protein, on the other hand, serves as a metal-binding partner that aids in the provision of nickel to the system.

Experiments

Stage 1:
Due to the excessive length of the highly efficient urease gene in S. pasteurii, we have divided it into two segments and cloned them onto corresponding plasmids separately. Specifically, UreA, UreB, and UreC are cloned onto the pET28a plasmid (with a T7 promoter), while UreE, UreF, UreG, and UreD are cloned onto the pET21b plasmid (with a proD promoter).(This page shows only the UreEFGD part)

Fig. 1 UreE, UreF, UreG,UreDcloned onto the pET21b plasmid (with a proD promoter)).

However, during the preparation of the urease activity detection medium and quantitative detection, the expression of urease was not apparent. Additionally, in subsequent SDS-PAGE analysis, the protein bands were also not distinct. Therefore, we further optimized our dual-plasmid system.

Fig. 2 SDS-PAGE result of stage 1.

Stage 2:
Based on the issues encountered during Stage 1 of urease expression, we plan to make the following improvements:
Change the promoter: Replace the proD promoter preceding UreE, UreF, UreG, and UreD with the strong T7 promoter to enhance the expression of downstream genes. Concurrently, we need to optimize the induction conditions, which primarily include the concentrations of IPTG and Ni²⁺, as well as the induction temperature and time, to improve the activity of expressed urease.
To achieve our goals, we will utilize the lab's plasmid pet21a (which contains the T7 promoter). We will design primers named vector-for, vector-rev, fragment-for, and fragment-rev. Next, we will perform PCR and inverse PCR to linearize the target genes and the vector, respectively. Following this, we will proceed with Gibson assembly to reconstruct the plasmid.

Fig. 3 UreE, UreF, UreG and UreD cloned onto the pET28a plasmid (with a T7 promoter)).

The BB fragment represents our final vector, pET21a, with a band length of 5310 bp after reverse PCR, while the INSERT corresponds to the target gene UreE-UreF-UreG-UreD, with a band length of 2698 bp after PCR. Running a nucleic acid gel confirmed that the observed band lengths matched the expected values.

Fig. 4 PCR Analysis of pET21a Gibson Assembly Reconstructed Plasmid on Nucleic Acid Gel (where BB: 5310 bp; INSERT: 2698 bp).

After gel extraction and purification of genomic DNA, we performed Gibson assembly to reconstruct the plasmid. The assembled DNA was then transformed into host cells for amplification and colony PCR screening. Ultimately, we retained the colonies with favorable sequencing results for plasmid extraction and dual plasmid transformation.

Fig. 5 pET21a transformation.

Similarly, this time we also examined the effect of urease expression through quantitative urease detection and SDS-PAGE analysis. The SDS-PAGE result is shown in Fig.4, while the principle and results of the quantitative urease detection are presented in Fig.5.The urease activity is 0.153U/g DCW（U：the amount of enzyme that catalyzes the formation of 1.0 µmole of ammonia per minute at pH 7.0.）

Fig. 6 SDS-PAGE result of stage 2.

Fig. 7 The principle and results of the quantitative urease detection.

Through the above experimental investigations, we have improved the recombinant expression of urease, and measurements have confirmed that the recombinant urease is active.
Stage 3:Mineralization Validation Experiment
We conducted a series of validation experiments, and some of the more representative results are as follows:
1.Optical Microscope Observation
As can be seen from Fig.6, compared to the BL21(DE3) control group, the dual-plasmid experimental group with recombinant urease exhibits significant differences in the mineralization of four metal ions: Li, Mn, Co, and Ni, under optical microscopy. Specifically, the bacteria in the experimental group show noticeable agglomeration and clustering, and a layer of material is clearly attached around the bacteria.

Fig. 8 Optical Microscope Observation of Biomineralization Results.

2.Atomic Force Microscopy (AFM) Observation

Fig. 9 Results of Biomineralization Observed by AFM. a) Dual-plasmid experimental group for Li;b) Dual-plasmid experimental group for Mn;c) Dual-plasmid experimental group for Ni;d) Dual-plasmid experimental group for Co;e) BL21 control group.

It can be observed that the bacteria in the control group are relatively dispersed, and the surrounding area appears flat. In contrast, the dual-plasmid experimental groups predominantly show aggregation of the bacteria, with noticeable elevation around them. AFM analysis confirms that a layer of material is indeed attached to the bacteria.
3.SEM/EDS analysis
Note: Due to the limitations of scanning electron microscopy, elements with an atomic number below that of carbon cannot be analyzed using EDS; therefore, Li cannot be detected with EDS.

Fig. 10 Electron Microscopy Images of Lithium, Manganese, Nickel, and Cobalt Carbonate Biomineralization(Note: The negative control uses wild-type BL21 bacteria co-cultured with metal ions without urease for biomineralization; the positive control involves the addition of urease to wild-type BL21 bacteria to simulate in vitro biomineralization; the experimental group uses BL21 bacteria expressing recombinant urease with dual plasmids for biomineralization.)a) Li negative control electron microscopy image;b) Mn negative control electron microscopy image;c) Ni negative control electron microscopy image;d) Co negative control electron microscopy image;e) Li experimental group electron microscopy image;f) Mn experimental group electron microscopy image;g) Ni experimental group electron microscopy image;h) Co experimental group electron microscopy image;i) Li positive control electron microscopy image;j) Mn positive control electron microscopy image;k) Ni positive control electron microscopy image;l) Co positive control electron microscopy image.

As shown in the figure above, the surfaces of the Li, Mn, Ni, and Co negative control bacteria are relatively smooth. The Li experimental group and positive control group exhibit swelling at one or both ends of the bacteria. In the Mn experimental group, the bacteria show swelling at one end, with a ring-like swelling in the middle and additional swelling at both ends; in contrast, the positive control also forms two hemispherical carbonate precipitates around the bacteria. The Ni experimental group presents a swollen appearance at one end and a swelling in the middle of the bacteria, similar to the positive control. The Co experimental group primarily displays a swollen appearance that resembles a club shape at one end and a swelling in the middle of the bacterial surface, while the positive control group shows no significant mineralization on the surface of the bacteria. Next, we will perform EDS analysis on the Mn, Ni, and Co groups to further validate the metal adsorption and mineralization capabilities of our recombinant urease-expressing Escherichia coli.

Fig. 11 EDS analysis of Mn. a)Schematic diagram of Mn carbonate biomineralization;b)EDS layered images of Mn;c)EDS analysis of Mn negative control;d)EDS analysis of the Mn experimental group;e)EDS analysis of Mn positive control.

The EDS resul

Fig. 12 EDS analysis of Ni. a)Schematic diagram of Ni carbonate biomineralization;b)EDS layered images of Ni;c)EDS analysis of Ni negative control;d)EDS analysis of the Ni experimental group;e)EDS analysis of Ni positive control.

Fig. 13 EDS analysis of Co. a)Schematic diagram of Co carbonate biomineralization b)EDS layered images of Co; c)EDS analysis of Co negative control;d)EDS analysis of the Co experimental group;e)EDS analysis of Co positive control.

The EDS results indicate that the elemental content analysis of Mn, Ni, and Co follows the trend: positive control > experimental group > negative control. This result is normal and relatively ideal, as the positive control group involved the direct addition of urease, allowing metal ions to interact with urease and decompose urea, generating carbonate ions that precipitate as carbonates. Consequently, the metal element content is highest in this group. The recombinant urease Escherichia coli adsorbs and mineralizes metal ions via its expressed urease. However, due to lower urease activity compared to that in the positive control group, the metal element content in the experimental group is slightly lower than in the positive control but significantly higher than in the negative control group. The negative control group, consisting of wild-type BL21 bacteria, does not express urease, rendering it incapable of biomineralization, resulting in the lowest metal element content.
In conjunction with the previously obtained SEM biomineralization images of Li, Mn, Ni, and Co, these findings effectively demonstrate that our recombinant urease-expressing Escherichia coli can successfully facilitate mineralization.

Conclusion

The results obtained from this study preliminarily demonstrate that the urease from S. pasteurii can be successfully expressed in Escherichia coli and can effectively facilitate mineralization. However, there remains considerable scope for improvement, particularly in enhancing urease activity and optimizing mineralization efficiency. Moving forward, our team will continue to refine the expression system and mineralization conditions, explore more efficient biomineralization methods, and strive to expand the application range of the resulting mineralization products, thereby contributing significantly to environmental protection and resource utilization.

Reference

[1]Fang L, Niu Q, Cheng L, et al. Ca-mediated alleviation of Cd²⁺ induced toxicity and improved ^Cd2+ biomineralization by Sporosarcina pasteurii[J]. Science of The Total Environment, 2021, 787: 147627.（https://www.sciencedirect.com/science/article/pii/S004896972102698X）
[2] Li M, Cheng X, Guo H. Heavy metal removal by biomineralization of urease producing bacteria isolated from soil[J]. International Biodeterioration & Biodegradation, 2013, 76: 81-85.（https://www.sciencedirect.com/science/article/pii/S0964830512001497）

Sequence And Features

Assembly Compatibility: