Part:BBa_K5166065
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)
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.
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.
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.）
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.
2.SEM/EDS analysis
Since the biomineralization of calcium carbonate is the most prevalent, we first used the biomineralization of Ca2+ as an example to verify the mineralization level of the recombinant urease. For the SEM and EDS analysis of Mn, Co, and Ni, please refer to our wiki result interface (due to the limitations of scanning electron microscopy, EDS cannot analyze elements lighter than carbon, so Li cannot be analyzed by EDS).
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.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.https://www.sciencedirect.com/science/article/pii/S0964830512001497）
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2448
Illegal XbaI site found at 47
Illegal XbaI site found at 927 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2448
Illegal NheI site found at 1415 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2448
Illegal BglII site found at 1947
Illegal BamHI site found at 73
Illegal XhoI site found at 712 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2448
Illegal XbaI site found at 47
Illegal XbaI site found at 927 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2448
Illegal XbaI site found at 47
Illegal XbaI site found at 927
Illegal AgeI site found at 1216
Illegal AgeI site found at 1379 - 1000COMPATIBLE WITH RFC[1000]
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