Part:BBa_M36071
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.
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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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.
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).
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.
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
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
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 346
Illegal PstI site found at 283
Illegal PstI site found at 593 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 346
Illegal PstI site found at 283
Illegal PstI site found at 593 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 346
Illegal XhoI site found at 43
Illegal XhoI site found at 127 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 346
Illegal PstI site found at 283
Illegal PstI site found at 593 - 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]
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