Difference between revisions of "Part:BBa K3523005"

Revision as of 16:47, 26 October 2020

T7 pro-His-SOD-His-T7 ter

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.

T7 pro-His-SOD-His-T7 ter

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

Improvement

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 (Fig 4). Apart from that, we add a T7 promoter, His tag, and a T7 terminator to form our composite part BBa_K3523005.

Figure 4 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 shows, 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 excellently degrade ROS into H2O2 that is in accord with our initial expectation.