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Revision as of 14:14, 18 October 2021

SOD

The excessive generation of reactive oxygen species (ROS) is considered to be one of the pathogenesis of IBD, and the use of SOD can significantly reduce the peroxide reaction in the inflected colon. Studies have shown that the expression level of Cu/Zn-SOD is decreased in IBD patients, and SOD cannot be directly treated, requiring carrier delivery. Therefore, we selected for heteroexpression of superoxide dismutase (SOD) gene from Lactobacillus casei Lc18.

Sequence and Features

Data:SZU-China 2021 TEAM

Anti-oxidative stress state of torsion (SOD) :

1. The DNA level

Two plasmids, PJSG(constitutive express SOD gene heat shock protein with GST label and triclosan resistance gene, ampicillin resistance in PUC18) and PET-S(constitutive exosomes express SOD gene with GST tag and His tag, kana-resistance in pet22b(+)), were selected for verification.

We extracted the target plasmid and amplified the SOD expression element on the plasmid, which was verified by DNA gel electrophoresis. We also carried out enzyme digestion verification on the plasmid, adding restriction endonuclease MIuI and SacI for double enzyme digestion, and obtained bands of the expected size, as shown in Figure 1. This further indicates that our plasmid transformation is successful.

The PET-S plasmid is an optimization of PJSG at a later stage of the experiment, removing two unnecessary genes, heat shock protein and triclosan resistance gene. We transformed the PJSG plasmid into DH5a and Nissle 1917, and peT-S into BL21(DE3). Among them, the transformation results of PJSG plasmid have been described above, as shown in figure 1. Plasmid extraction was carried out for transformed BL21(DE3) and double enzyme digestion was performed with XhoI and NcoI, as shown in figure 2.

Figure 1 A. Gel electrophoresis of DNA transformed into EScherichia coli DH5α by plasmid PJSG (5939 bp). B.After transformation, Nissle 1917 SOD expression element was amplified(target fragment size was 1890 bp) c. The PJSG was double-digested with restriction endonuclease MIuI and SacI, and the expected band sizes were 1918 bp and 4021 bp

Figure 2 Enzyme digestion of PET-S plasmid extraction

2. Protein level

We then carried out protein level verification on the engineered bacteria. As shown in Figure 3A, for DH5α, compared with the blank group in lane 1, as indicated by the arrow, SOD (with GST label) protein band of 55.14kDa could be seen in lane 2 and SOD (with 6x His label) protein band of 29.5 kDa could be seen in lane 3, which were clear and met the expected size. As shown in figure 3B, for Nissle 1917, compared with the blank group in lane 1, as indicated by the arrow, the SOD (with GST label) protein band of 55.14 kDa could be obviously seen in lane 2, and the SOD (with 6x His label) protein band of 29.5 kDa could be obviously seen in lane 3, with clear bands and sizes meeting expectations.

Since our plasmid PJSG is expressed in constitutive exogenesis, we performed SDS-PAGE on cells culture supernatant to detect its extracellular proteins. Unfortunately, DH5α did not detect any exocrine expression, which may be related to its lack of strong protein expression ability. However, we detected exotic SOD protein in the culture supernatant of transformed Nissle 1917, with the same band size as expected, as shown in figure 3C (red arrow)

Figure 3 A. Lane 1 is DH5α intracellular protein without plasmid transformation, lane 2 is DH5α intracellular protein transformed into PJSG plasmid, and lane 3 is DH5α intracellular protein B. transformed into PJSH plasmid. Lane 1 was Nissle 1917 intracellular protein without plasmid transformation, lane 2 was Nissle 1917 intracellular protein with plasmid transformation of PJSG C. Lane 1 was Nissle 1917 extracellular protein without plasmid transformation, lane 2 was Nissle 1917 extracellular protein with plasmid transformation of PJSG.

For BL21 transformed into PET-S, SDS-PAGE and WB detection were performed. It should be noted that in the plasmid design, we did not delete the induction expression system on the original PET-22b (+), but inserted the whole part including promoters, terminators, and coding sequences into the polyclonal enzyme restriction site region of PET-22b (+). Therefore, in theory, it can be continuously expressed under the control of constitutive promoter PJ23100 and also support IPTG induced expression. Through the difference of protein expression levels between induced and uninduced, it can be proved that this protein is expressed. SDS-PAGE as shown in figure 4A, the red arrow points to the expressed target protein. Subsequent WB results also showed that the protein was expressed, as shown in figure 3B.

Figure4 A. Lane PC1 is BSA(1μg). Lane PC2 is BSA(2μg). Lane NC is cell lysate without induction. Lane 1 is cell lysate with induction for 16h at 15℃. Lane 2 is cell lysate with induction for 4h at 37℃. Lane NC1 is a supernatant of cell lysate without induction. Lane 3 is supernatant of cell lysate with induction for 16h at 15 ℃. Lane 4 is supernatant of cell lysate with induction for 4h at 37℃. B. Lane 3 is supernatant of cell lysate with induction for 16h at 15 ℃. Lane 4 is supernatant of cell lysate with induction for 4h at 37℃. Lane 5: Pellet of cell lysate with induction for 16h at 15℃. Lane 6: Pellet of cell lysate with induction for 4h at 37℃.

3. Functional representation

We first measured the SOD activity of Nissle 1917 cell contents transformed with PJSG plasmid and blank Nissle 1917 cell contents without transformed plasmid as an internal reference. Unit enzyme activity was defined as one SOD activity unit (U) when the SOD inhibition rate reached 50% in the reaction system. Because the engineered bacterial transfer algebra used for each measurement was different, we treated each measurement as an independent experiment, so we made our own standard protein concentration curve for each batch of measurements. The batch standard curve and enzyme activity are shown in Figure 5. SOD activity in Nissle cell contents after transformation was 23.55938 U and 17.91151 U after the internal reference was deducted.

Figure 5.A standard curve of protein concentration B. Intracellular superoxide dismutase activity of Nissle 1917 after transformation Then we measured SOD activity in the supernatant of Nissle medium with transformed pJSG plasmid and in the blank Nissle medium without transformed plasmid as an internal reference to determine the effect of plasmid exosecretion expression. Meanwhile, SOD activity of DH5α cell contents transformed with pJSG plasmid and blank DH5α cell contents not transformed with pJSG plasmid were determined as an internal reference. The standard curve and enzyme activity of this batch are shown in Figure 6.

After transformation, SOD activity was 3.490339 U in the supernatant of Nissle 1917 medium, and 1.475081 U in the supernatant of Nissle 1917 medium after deduction of internal reference, indicating low secretion and expression efficiency. It was considered that the culture conditions were not suitable for secretion and expression and the target protein was secreted into the periplasmic space. SOD activity of the transformed DH5α cell contents was 41.35685 U, and 12.40467 U after deducting internal reference. Under the same conditions, the expression efficiency of Nissle 1917 heterologous superoxide dismutase was higher than that of DH5α.

Figure 6 A. BSA standard curve B. Superoxide dismutase activity of transformed Nissle 1917 exosecretion C. Intracellular superoxide dismutase activity of DH5α after transformation

SOD activity of BL21(DE3) cell contents transformed with PET-S plasmid and blank BL21(DE3) cell contents without transformed plasmid were again used as internal references. At the same time, the activity of the SOD enzyme in the supernatant of its culture medium was also determined. The batch standard curve and enzyme activity are shown in Figure 7. The intracellular protease activity of BL21(DE3) after transformation was 404.6549 U, and 176.7827 U after deducting internal reference. After transformation, SOD activity in the supernatant of BL21(DE3) medium was 132.1789 U, and after deducting internal reference, SOD activity was 111.7859 U. These results indicated that the plasmid had a strong expression ability in BL21(DE3) and could be successfully expressed by exogenesis. Thus, we measured the enzyme activity-time curves as follows, which meet the general enzymological characteristics.

Figure 7A. BSA standard curve b. Intracellular and extracellular superoxide dismutase activity of transformed BL21(DE3) C. After deducting internal parameters, the change curve of intracellular SOD activity expressed by transformed BL21(DE3) with time D. After deducting internal parameters, the change curve of SOD enzyme activity expressed in the transformed BL21 (DE3) exoskeleton with time.

We hope to use BL21(DE3) which had transformed PET-S plasmid to further determine and explore the enzymatic characteristics of the allogeneic SOD, especially the enzymatic temperature curve, to prove that our enzyme is suitable for the temperature environment in the intestinal tract. We made standard curves and enzyme activity-temperature curves respectively, as shown in figure 8.

The results showed that 37℃ was the optimum temperature for enzyme activity. In addition, the proteins from the enzyme activity-time curve and enzyme activity-temperature curve were not made in the same batch, and the bacteria were not in the same batch. The bacteria of the latter are obtained through multiple rounds of the former, so there may be degradation involved, and other experimental operation problems resulting in the enzyme activity of the latter is not as high as the former, but it is still meaningful.

Figure 8A. Protein standard curve