Difference between revisions of "Part:BBa M36071"
(→‎Improved:SZU-China 2021:The addition of signal peptides)
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[[File:T--SZU-China-BBa K3838888-SOD1.png|400px|thumb|center|Fig6.Enzyme activity time curve of SOD cells]]
 
[[File:T--SZU-China-BBa K3838888-SOD1.png|400px|thumb|center|Fig6.Enzyme activity time curve of SOD cells]]
 
[[File:T--SZU-China-BBa K3838888-SOD3.png|400px|thumb|center|Fig7.Time curve of SOD extracellular enzyme activity]]
 
[[File:T--SZU-China-BBa K3838888-SOD3.png|400px|thumb|center|Fig7.Time curve of SOD extracellular enzyme activity]]
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More data of this improved part are as follows:
 +
Anti-oxidative stress state of torsion (SOD) :
 +
 +
1. The DNA level
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 +
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.
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[[File:T--SZU-China--resultfigure9.png|500px|center]]
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<center>Fig.1 Enzyme digestion of PET-S plasmid extraction.</center>
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2. Protein level
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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)
 +
 +
[[File:T--SZU-China--resultfigure10A.png|500px|center]]
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[[File:T--SZU-China--resultfigure10B.png|500px|center]]
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[[File:T--SZU-China--resultfigure10C.png|500px|center]]
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<center>Fig.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.</center>
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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 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.
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[[File:T--SZU-China--resultfigure11A.png|500px|center]]
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[[File:T--SZU-China--resultfigure11B.png|500px|center]]
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<center>Fig.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 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℃.</center>
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3. Functional representation
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We first measured 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 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 the internal reference was deducted.
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[[File:T--SZU-China--engineeringsuccessfigure2A.png|400px|center]]
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[[File:T--SZU-China--ES3.png|500px|center]]
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<center>Fig.4 A standard curve of protein concentration. B Intracellular superoxide dismutase activity of Nissle 1917 after transformation.</center>
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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 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.
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 +
[[File:T--SZU-China--measurementfigure3A.png|400px|center]]
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[[File:T--SZU-China--ES5.png|500px|center]]
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[[File:T--SZU-China--ES6.png|500px|center]]
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<center>Fig.5 A BSA standard curve. B Superoxide dismutase activity of transformed Nissle 1917 exosecretion. C Intracellular superoxide dismutase activity of DH5α after transformation.</center>

Revision as of 17:27, 21 October 2021

K12 e.coli native Manganese Superoxide Dismutase

Coding sequence for the enzyme Mn-SOD or SoDA. This protein catalyzes the dismutation of superoxide, a free radical. Sequence found by converting the amino acid sequence of Mn-SOD protein native to Escherichia coli, the organism for which it is also optimized for.


Usage and Biology

Superoxide dismutase cataylyzes the dismutation of superoxide. Superoxide is produced naturally in the respiration process. It causes damage to the cell by altering lipid structure and breaking down proteins among other negative effects. This gene provides the protein to transform two superoxide molecules into hydrogen peroxide and oxygen.

Design Notes

This DNA sequence is converted from the amino acid sequence of Mn-SOD protein using the online converter from In-Silico. We optimized our sequence in DNA 2.0's Gene Designer by increasing GC content and minimizing repeats. We accomplished this by altering basepairs without altering amino acids.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 346
    Illegal PstI site found at 283
    Illegal PstI site found at 593
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 346
    Illegal PstI site found at 283
    Illegal PstI site found at 593
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 346
    Illegal XhoI site found at 43
    Illegal XhoI site found at 127
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 346
    Illegal PstI site found at 283
    Illegal PstI site found at 593
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 346
    Illegal PstI site found at 283
    Illegal PstI site found at 593
    Illegal NgoMIV site found at 542
  • 1000
    COMPATIBLE WITH RFC[1000]



Improvement Made by Xiamen_city 2020

Our composite part BBa_K3523005 contains the basic part BBa_K3523000, which sequence has optimized differently from the sequence of other two similar basic part BBa_M36071 and BBa_K2638106 (Fig1). Apart from that, we add a T7 promoter, His tag, and a T7 terminator to develop our composite part BBa_K3523005.

Fig1.A sequence assay of the three basic parts BBa_K3523000, BBa_M36071 and BBa_K2638106.

There is a mini-review of the development of SOD protein related parts. In 2012, group Stanford BIOE44-S11 designed a basic part BBa_M36071, aimed to catalyze the dismutation of superoxide. Although they attempted to utilize and overexpress this protein, they did not get their expected results. In 2018, group iGEM18_Bielefeld-CeBiTec designed a basic part BBa_K2638106, which has optimized the coding sequence of SOD protein. They also designed some related composite parts, like BBa_K2638117, BBa_K2638118, and BBa_K2638116. However, all these parts did not attach any experimental data to prove the function of SOD protein. Today, our team further improved the coding sequence of SOD protein and constructed composite part BBa_K3523005. In order to prove the function of these parts, we expressed and purified SOD protein, and then detected the enzyme activity in vitro. As the result shown, our SOD protein has achieved engineering success. Besides, our project aimed to degrade reactive oxygen species (ROS) accumulated when people staying up late. And the SOD can excellent degrade ROS into H2O2 that is accord with our initial expectation.

BBa_K3523005 contains BBa_K3523000, encoding the superoxide dismutases (SOD). SOD is a group of enzymes that catalyze the dismutation of superoxide radicals (O2−) to molecular oxygen (O2) and hydrogen peroxide (H2O2), providing cellular defense against reactive oxygen species.

Contribution and Biology

Our goal of this project is to construct an engineered bacteria which will scavenge superoxide compounds (ROS) in gut quickly and efficiently. To achieve it, we selected a classical enzymes -- superoxide dismutase (SOD), which are capable of effectively degrading ROS, for overexpression and purification in Escherichia coli BL21 (DE3). By monitoring ROS consumption, the ability of the engineered strain to degrade ROS was verified.

Figure 1

We use T7 promoter to start SOD protein transcription, and T7 terminator to end transcription. At the same time, insert a His protein tag into SOD protein for purification of SOD protein on the nickel column. This part can be used for topics related to the degradation of ROS in the future.

Engineering Success

Characterization of the biochemical characteristics of SOD:

SOD was expressed in Escherichia coli, bacterial cells were collected and broken, and SOD solution was obtained through isolation and purification, and further confirmed by the SDS-Page method, protein bands of the corresponding size were found (Fig.2).

Figure 2 SDS-Page assay the expression of SOD protein M: Protein Ladder; FT: Flow-through sample; W: Washing sample; 50: Elution sample with 50mM imidazole; 100: Elution sample with 100mM imidazole; 250: Elution sample with 250mM imidazole; 500: Elution sample with 500mM imidazole.


We used the classic nitroblue tetrazolium (NBT) color development method. Superoxide anion (O2-.) was produced by Xanthine and Xanthine Oxidase (XO) reaction system to reduce NBT to blue formazan, which had strong absorption at 560nm. While SOD can remove superoxide anions, so dirty formation is inhibited. The bluer the reaction solution is, the lower the activity of superoxide dismutase is, and vice versa. The activity level of superoxide dismutase can be calculated by colorimetric analysis. The detection principle is shown in Fig.3, and the detected absorbance is shown in Table.1.

Figure 2
Figure 3

The data is substituted into the formula for calculation:

Inhibition percentage=[(Ablank1-Ablank2) - (Asample-Ablank3)]/(Ablank1-Ablank2) * 100%=69.543%

Enzyme activity of sample=inhibition percentage / (1-inhibition percentage) (units)=2.283 U

Specific activity of SOD= enzyme activity of sample / amount of protein (units/mg)=1936.12 U/mg.

The results showed that SOD protein became dissolved in this E. coli expressing a condition, and the target protein is very pure. And SOD had excellent catalytic properties, which could successfully degrade ROS into H2O2

Improved:SZU-China 2021:The addition of signal peptides

SZU-China has improved the existing part by adding the signal peptide.

Fig4.The design of the improved part

SOD activity of BL21(DE3) cell contents transformed with PET-S plasmid was used. At the same time, the activity of SOD enzyme in the supernatant of its culture medium was also determined.

Enzyme activity As shown in Figure 5, the enzyme activity of BL21(DE3) intracellular protein after transformation was 176.7827U except for internal reference, and the enzyme activity of BL21(DE3) culture medium after transformation was 111.7859U after SOD subtracting internal reference, which indicated that this plasmid had strong expression ability in BL21(DE3) and could successfully express exogenesis. Thus, we measured the enzyme activity-time curves,Figure 6、7, which meet the general enzymological characteristics.

Fig5.SOD activity in and out of cells
Fig6.Enzyme activity time curve of SOD cells
Fig7.Time curve of SOD extracellular enzyme activity

More data of this improved part are as follows: 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.

T--SZU-China--resultfigure9.png
Fig.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)

T--SZU-China--resultfigure10A.png
T--SZU-China--resultfigure10B.png
T--SZU-China--resultfigure10C.png
Fig.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 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 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.

T--SZU-China--resultfigure11A.png
T--SZU-China--resultfigure11B.png
Fig.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 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 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 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 the internal reference was deducted.


T--SZU-China--engineeringsuccessfigure2A.png
T--SZU-China--ES3.png
Fig.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 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 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.

T--SZU-China--measurementfigure3A.png
T--SZU-China--ES5.png
T--SZU-China--ES6.png
Fig.5 A BSA standard curve. B Superoxide dismutase activity of transformed Nissle 1917 exosecretion. C Intracellular superoxide dismutase activity of DH5α after transformation.