Part:BBa_K4644010

pETlac-ureABC-lac-ureEFGD

        Urea degradation module-The second expression frame we constructed.

Biology

        Urease is an enzyme that catalyses urea hydrolysis, forming carbon dioxide and ammonia. In biomineralization, carbonic anhydrase acts as a hydrator, catalysing the reaction of carbon dioxide (CO2) with water into carbonic acid, which spontaneously decays into carbonate ions.Bacterial urease genes were widely reported in a way of urease gene cluster, which was consisted of structural genes and accessory genes. Structural subunits of urease were encoded by structural genes (ureABC), and accessory subunits of urease were encoded by accessory genes (ureEFGD).The accessory genes (ureDEFG) are involved in the incorporation of nickel ions into the apoenzyme of urease.

Fig 1. Degradation of urea

        Using pETlac-ureABC and pETlac-ureEFGD as templates, we amplified the two expression frames separately. After connecting them, we homologously recombined them with the pETlac plasmid backbone to obtain this.

Fig 2. pETlac-ureABC-lac-ureEFGD

Usage

Plasmid Amplification and Verification

        We will amplify the constructed plasmid by chemical transformation in E. coli DH5α and then spread it on ampicillin-resistant solid LB agar plates for resistance selection. Positive colonies will be selected and subjected to colony PCR screening (Figure 3, using ureABC as PCR primers) to obtain strains carrying the correct plasmid.

        These strains will be cultured in liquid LB medium supplemented with ampicillin resistance at 37°C with shaking overnight. Subsequently, plasmids will be extracted to ensure the correctness of the plasmid sequence. They will be sent to a gene company for sequencing to confirm their accuracy.

Fig 3. pETlac-ureABC-lac-ureEFGD Colony PCR Results（right）

Soluble Expression of Urease

        In E. coli expression systems, inclusion bodies can form when expressing foreign proteins due to the lack of modification systems. Currently, the mainstream approach in the scientific community to increase solubility is to purify and refold the expressed proteins or use molecular chaperones or solubilization tags. Therefore, BUCT has attempted four molecular chaperones to enhance solubility: GroEL/GroES, DnaK/DnaJ, IbpAB, and sigma32. The plasmid maps for each of these chaperones are shown in the figure below.

Fig 4. pcs-lac-grosl

Fig 5. pcs-lac-dnaKJ

Fig 6. pCS-lac-sigma32

Fig 7. pCS-lac-ibpAB

Solubility Verification:

        The gene segments constructed as described above were subjected to homologous recombination with the pET-duet plasmid using the same method. E. coli BL21 cells, which had been pre-cultured and stored, were revived. The plasmids were transformed into the host cells through electroporation. Four different molecular chaperones were separately introduced into the host cells, and a control group with an empty vector and another control group with no molecular chaperones were included, resulting in a total of six groups.After selection for antibiotic resistance, the cells were initially inoculated into LB medium containing both ampicillin and kanamycin antibiotics.

        Subsequently, they were transferred to 50 ml of LB medium containing both antibiotics in shake flasks and cultured at 37°C until the OD reached the desired range (0.2-0.6). The cultures were induced with 0.5 mM IPTG and further incubated for 12-16 hours.The fermentation broth was then collected and subjected to repeated sonication for cell disruption. Each group was divided into two fractions: the supernatant and the precipitate.         Finally, the gel electrophoresis results for this protein were obtained using SDS-PAGE.

Fig 8. Urease SDS-PAGE-(1)

Fig 9. Urease SDS-PAGE-(2)

After comparison, it is easy to notice that the effect of GroEL/GroES

‘’‘Ability of degrading urea’‘’

        Set Control and experimental groups.Samples of the fermentation broth are taken at 12, 24, 36, and 48 hours of fermentation. The samples are processed and analyzed using High-Performance Liquid Chromatography (HPLC) to obtain fermentation data for this module. Due to the inherent metabolic activities of Escherichia coli, it is difficult to measure the exact amount of ammonia produced. However, fortunately, E. coli itself cannot directly metabolize urea. Therefore, the decrease in urea concentration can directly reflect the performance of this module.

table 1. Urea consumption in the experimental group

table 2. Urea consumption in the control group

Sequence and Features

Assembly Compatibility:

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