Part:BBa_K3523005

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.

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

Characterization by 2021iGEM_Shanghai_Metro_Utd

Improvement of an existing part

Figure 9. The blast results about the DNA sequence of our new part BBa_K4005004 and the old part BBa_K3523005..

Compared to the old part BBa_K3523005, composite part T7 pro-His-SOD-His-T7 ter, we design a new part BBa_K4005004, which replaced the SOD gene sequence fragment with rANG gene fragment. According to literature reports, heparan sulphate proteoglycans (HSPGs) are required for ANG to enter cells. HSPGs are linear anionic glycosaminoglycan (GAG) chains covalently linked to the core protein, composed of disaccharide repeating units of uronic acid and glucosamine, and the glucosamine residues 3-OH, 6-OH and -NH And the 2-OH of uronic acid is replaced by a sulfate group. HSPGs exist on the plasma membrane of all animals and are the main component of the extracellular matrix. Factors such as size, location, degree of sulfation, and uronic acid in different cells, tissues, and developmental stages contribute to the structural diversity of heparan sulphate (HS). This structural diversity is the basis for HSPGs to perform multiple functions. In previous research, our laboratory constructed a library of HS mutant mouse lung endothelial cells by knocking out key genes in the HS biosynthesis process. In this study, we prokaryotic expression and purification of recombinant ANG protein, using heparin and heparin derivatives and HS mutant mouse lung endothelial cell library to perform enzyme-linked immunosorbent assay and flow cytometry experiments to explore the interaction between ANG and HS The specificity and clarify its structure-activity relationship.

Usage and Biology

Angiogenin (Angiogenin, ANG) belongs to the secreted ribonuclease superfamily, and was originally discovered to be an angiogenic factor derived from human tumors. Subsequent studies have shown that ANG is also present in normal human tissues and body fluids. ANG increases the transcription of ribosomal RNA (rRNA) in the nucleus, while vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) activate mTOR and S6 kinase pathways to promote protein synthesis. Therefore, ANG can cooperate with VEGF and FGF-2 to promote protein synthesis in endothelial cells. In addition, ANG is necessary for endothelial cell proliferation. In addition to promoting angiogenesis, ANG is also involved in many other physiological and pathological processes, such as neuroprotection, inflammation, and tumorigenesis. Loss of function mutations in the ANG gene have been found in both amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD).

Construct design

Figure 1. The rANG protein expression box..

The profiles of every basic part are as follows:

BBa_K4005000

Name: rANG

Base Pairs: 375bp

Origin: Homo sapiens (Human)

Properties: A coding sequence for Angiogenin.

Usage and Biology

ANG is necessary for the proliferation of endothelial cells, and mutations in the loss of function of the ANG gene are likely to be important causes of cardiovascular diseases such as amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD)

BBa_K4005003

Name: 6His

Base Pairs: 18bp

Origin: synthetic

Properties: Polyhistidine tag

Usage and Biology

It is an polyhistidine tag, which is used in the purification of recombinant proteins

BBa_K4005001

Name: P7

Base Pairs: 19bp

Origin: T7 phage, genome

Properties: A promoter for initiation of the transcription.

BBa_K4030002

Name: TT

Base Pairs: 140bp

Origin: Escherichia coli

Properties: Transcription terminator

Usage and Biology

It is an transcription terminator derived from the E.coli rrB rRNA operon

Experimental approach

pET-28a-rANG which produces ANG protein is shown in Figure 1.

Figure 2. Schematic map of pET-28a-rANG plasmid and its construction..

Figure 3: the scanning gel electrophoresis map of the plasmid vector after enzyme digestion.

Figure 3: the scanning gel electrophoresis map of the plasmid vector after enzyme digestion.

As seen in figure 3, lane 1 is the pET-28a without enzyme digestion and lane 2 to 9 is the result for pET-28a after enzyme digestion. The bands of our plasmid vectors after enzyme digestion(5311bp) showed at 5000 bp around which are correct and the bands at 300 bp around were the SUMO fragments that were cut by enzyme digestion. After that, we could further conduct the E. coli transformation.

Figure 4: The gel electrophoresis map of PCR.

Lane 1 to 6 is the result of PCR. We got rANG band at around 400bp (369bp). Enzyme digestion was conducted and it was linked with digested pET-28a.

Figure 5. E. coil having the desired pET28a-rANG.

Figure 5. E. coil having the desired pET28a-rANG.

The plates in Fig. 5 (1) showed monoclonals of pET28a-rANG constructs. The pET28a-rANG was constructed successfully which has been proved by sequencing as shown in Fig. 5 (2).

Recombination E. coli

Figure 6: the scanning gel electrophoresis map of Colony PCR (the top was before and the bottom was after).

Figure 6: the scanning gel electrophoresis map of Colony PCR (the top was before and the bottom was after).

Lane NC to XCN6 and lane XCN7 to N6 are the results of colony PCR. We get the band 531bp at 500bp around. It indicates that the obtained recombinant monoclonals were positive monoclonals containing the pET-28a-rANG recombinant plasmid.

Proof of function

We did SDS-PAGE assey of protein and have obtained Freeze-dried powder of the ANG protein produced by our engineered Escherichia coli (Figure 7).

Figure 7. SDS-PAGE assey of protein and Freeze-dried powder of the ANG protein..

Figure 7. SDS-PAGE assey of protein and Freeze-dried powder of the ANG protein..

The activity of recombinant ANG protein was determined by the RNA degradation assay. The result indicated that ANG protein possesses ribonuclease activity as we expected.

Figure 8. RNA degradation by ANG..

Future plan

In future applications, the product we get may have two directions of develop, but all in the clinical medicine aspect. One is to directly invent a new drug with the protein product of this gene and the other one is to explore its pharmacology regarding the relevant diseases which could be used to develop more new drugs.

The first kind of drug may have an effect on adjusting the nerve system, treating diseases like inflammation and the further developed drugs may be a possible treatment of some present incurable diseases such as PD and tumor. However, the overuse of ANG will also overpromote the generation of endothelial cells and vessels and may even cause cancer in some situations, so the correct usage of this drug shall be also necessary for being studied.

Market Survey According to relevant data, among the 65-year-olds, one in ten people are Parkinson's disease patients; among the 85-year-olds, the number of people who have Parkinson's disease reaches 30%-50%. The research progress and molecular biological characteristics of Angiopoietin and its related proteins are reviewed. So far, four kinds of Angiopoietin and some isoforms or variants of Angiopoietin have been discovered. These proteins have a common structure: a signal peptide related to secretion at the amino terminus, a helix-like domain that mediates homo-oligomers, and a fibrinogen-like domain that mediates ligand activity at the carboxyl terminus.

Marketing Plan The theory we have resulted in a unique product (but not medicine), which will be studied and researched by staff members in the collaborated pharmaceutical company), and the final medical products will be entirely produced and owned by the partner company. The outcome of our company remains with unknown effects on medicine. However, the result will be owned by our target group (pharmaceutical company).

We will market our product using many different ways. For example, online surveys, email marketing, mobile marketing, and social media marketing are considered the cheapest among all types of marketing. Since we are a brand new company, we do not have many budgets available for us, so we would consider the ways I listed above first. Once we have more budget allowed, we might try other ways of marketing our product. For example, printed marketing materials, such as brochures, flyers, business cards, etc. We can also try trade shows and other marketing that cost some money.

Challenges During the experimental work, the main challenges were the plasmid DNA extraction and the protein purification process. One aspect of the difficulty is the continuous low concentration of plasmid DNA when we first extracted them. But as we navigate through and become more familiar with the long process, we successfully extracted with an ideal concentration.

Moreover, heterologous overexpression often results in low expression levels and inactive protein due to insufficient membrane insertion and folding or lack of post-translational modifications. And over-expression of membrane proteins can be toxic to the cell. Nevertheless, eventually, we obtained the freeze-dried protein powder. But with no doubt, we will further solidify this part of the work.

Before our product come into a real application, there are still some concerns regarding our Angiogenin protein. We need to conduct more experiments and tests to further ensure its purity, stability and biological activity. In addition, we would further focus on the yield issues, storage conditions and how to use our products to determine the dose of curing some present incurable diseases.

Reference

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]