Part:BBa_K3838888

partinfoBBa_K3838888 shortpartinfo

The overproduction of Superoxide dismutase (SOD) and reactive oxygen species (ROS) is considered to be one of the pathogenesis of IBD. The use of SOD can significantly reduce the peroxidation reaction in the inflamed colon. Studies have shown that the decreased expression of CuZn-SOD has been observed in IBD patients. SOD can not be directly treated, so carrier transport is needed. Therefore, we selected for heteroexpression of superoxide dismutase (SOD) gene from Lactobacillus casei Lc18. In a team project, SOD related parts are composed as follows J23100 promoter +RBS+SOD+ HIS tag +Signal peptide+T7 terminator

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 394
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 394
  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 394
  Illegal BglII site found at 437
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 394
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 394
  Illegal NgoMIV site found at 598
  
• 1000
  COMPATIBLE WITH RFC[1000]

Data:SZU-China 2021 TEAM

Anti-oxidative stress state of torsion (SOD) :

1. The DNA level

Two plasmids, PJSG and PET-S, were selected for verification. 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 1.

Figure 1. 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 2A, 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 2B, 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 cell 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 2C (red arrow)

Figure 2 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 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 level between induced and uninduced, it can be proved that this protein is expressed. SDS-PAGE as shown in figure 3A, the red arrow points to the expressed target protein. Subsequent WB results also showed that the protein was expressed, as shown in figure 3B.

Figure 3 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 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 SOD activity of Nissle 1917 cell contents transformed with PJSG plasmid and blank Nissle 1917 cell contents without transformed plasmid as internal reference. Unit enzyme activity was defined as one SOD activity unit (U) when 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 4. SOD activity in Nissle cell contents after transformation was 23.55938 U and 17.91151 U after internal reference was deducted.

Figure 4.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 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 internal reference. The standard curve and enzyme activity of this batch are shown in figure 5.

After transformation, SOD activity was 3.490339 U in the supernatant of Nissle medium, and 1.475081 U in the supernatant of Nissle 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 5 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 SOD enzyme in the supernatant of its culture medium was also determined. The batch standard curve and enzyme activity are shown in figure 6. 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 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 6 A. 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.