The the selenite reducing ability of STaphylococcus aureus LZ-01 and Pseudomonas aeruginosa LZ-4 was verified in the laboratory, and E. Coli was used as control group. After resuscitation from the 4℃ refrigerator, cultures were inoculated at 1% (V/V) in 100 mL medium to prepare the same concentration of inoculum (OD600 = 1.0). The inoculum of the three strains was compared at 1% (V/V) in 37℃ in 5 mL LB medium (3 parallel groups) for 12 h, with 2mM Na2SeO3, and another 12 h. The supernatant was then collected and centrifuged to indentify the reduction rate by observing the amount of elementary red Selenium(8,000 rpm,4℃, 10 min). *The Na2SeO3, of 0.1729 g was dissolved in 1 mL deionized water and filtered using a sterile filter head (0.22 μm).
It was found that LZ-01 has the highest selenite reduction rate of 88.58%. For LZ-4 and E.Coil, the reduction rate of coli was relatively similar, with 45.25% and 51.53%, respectively. LZ-01 is 1.96 times more effective than LZ-4 and E.coli was 1.72-fold, with a significant difference (p < 0.01) . Therefore, we used LZ-01 as the target strain to explore its functional gene of selenite reduction.
For strain LZ-01, we used bioinformatics methods to compare the strain with selenite reducing proteins in NCBI database, and found three target genes of this experiment, namely SAV 0956, SAKG03 26900 and SAKOR 01018.
To validate the function of the three genes in Se (IV) reduction, three pairs of primers were designed to amplify the genes from LZ-01. After the fragments were digested with EcoRI and XhoI, the expressed protein was inserted into the plasmid pET-28a (+) by the ligation reaction. After purification by nickel column, we eluted and obtained the purified target protein. We adjusted the protein concentrations of the three parallel groups to 1 mM, adding the addition of 2 mM NADH and 0.5 mM sodium selenite in buffer of Tris-7.4. Then the protein reductiveness was verified by 30min in vitro reaction 30 min.
The reaction centrifuge tube from left to right is: control (no protein), SAKG03 26900, SAKOR01018, SAV 0956 expressed protein, it can be observed that the control group completely no red material, the most red material was presented by the protein of the SAV0956 gene expression. According to the examination, the SAV 0956 gene expressed the strongest protein reduction capacity, reducing 59.73% Se (IV) after 0.5 h, followed by 29.2% Se (IV) from SAKG03 26900.
Thus, we selected the SAV0956 gene as the functional gene and named the selenite-reducing protein it expressed as SerV01. Compared with other selenite reductases reported, SerV01 enzyme can achieve In vitro purification validation directly. It reduces selenite without the cooperation of multiple enzymes, which saves costs and has high stability. Hence, SerV01 enzyme would be the ideal option.
We then examined the effect of the environment(PH and temperature) on protein reduction efficiency after purification, using NADH as an electron donor. It was detected that the degradation rate reached the maximum at 8.0, and Na2SeO3 reduction efficiency gradually decreased when pH was greater than 8.0. Futhermore, we examined show that the enzymatic reaction is almost inactive at 4 °C, and the reduction rate of Na2SeO3 gradually increases, with the highest reduction efficiency of Na2SeO3 when the temperature conditions reach 37 °C. Therefore, the reaction optimal temperature was 37 °C, with an optimal pH of 8.0 while the simulated animal gut environment is about pH = 7.0 at 37℃. Hence, we speculated that the enzyme can maintain high activity in gut environment, which is the proposed implementation of us.
The SerV01 protein in LZ-01 was successfully anchored to the extracellular membrane of probiotics EcN using cell surface display technique in this part. Firstly, carrier protein INP was obtained, and its N-terminal domain was used as the template to design primers. SerV01 sequence was selected as the expressed protein to design the primers copy gene fragment, and used to design the forward and backward primers, serv-F and Ser-R, of Overlap PCR. To make the fusion protein express more in the following process, we added strong promoter and long gap sequence. After comparing the iGEM library, the high-copy plasmid pSB1A3 was obtained. By using homologous recombination method to do the cloning and transformation after overlap PCR amplification, the INP-SerV01 was linked together with plasmid pSB1A3. Blunting Kination Ligation (BKL) Kit first linearized its circular DNA molecules and subsequently incubated with the target fragment. Recombinant plasmid containing the INP-SerV01 fusion polypeptide was named pSB1A3-INP-SerV01. The pSB1A3-INP-SerV01 plasmid was first transformed to the E.In coli DH5α competent cells. The correct recombinant plasmid in Coli DH5α was taken and converted to EcN for clonons in the same manner described above. Among these, E. coli containing the plasmid pSB1A3-INP-SerV01 was named EcN-IS. After expression, INP-SERV01 fusion peptide was formed on the cell surface.
Wild-type EcN and EcN containing only empty-load pSB1A3 were used as control strains to do the expression examination. To validate differences in the reaction reduction efficiency of the experimental group of engineered bacteria and only empty plasmids, the residue of tetravalent selenium in the supernatant was detected at 4,8,12 and 24 h sampling, respectively, as shown from the figures, little residue of tetravalent selenium was detected in the supernatant at culture to 24h, indicating termination of the reaction. It shows that the selenite residues at 8th h, EcN-IS and EcN-pMD1A3 are 67.9% and 57.8%, respectively. Similarly, the selenite residues at the 12th h, EcN-IS and EcN-pSB1A3 were 46.7% and 57.8%, respectively. Therefore, the average reduction capacity of EcN-IS is about 10% higher than that of EcN-pSB1A3 (P< 0.05), indicating that constructing selenite reductase on the surface can have a faster reduction efficacy. Figure b shows the bacterial growth curve, and the strain concentration remains similar, but the reduction rate varies, further indicating that the surface display enzymes can play a certain promoting effect. The main reason may be to reduce the time of selenite into E. coli and the cross cell membrane after reduction, followed by reducing the burden on bacterial cells, so the bacterial growth is more stable and does not lead to inefficient reduction due to excessive selenotoxicity.
