Difference between revisions of "Part:BBa K1898200"

Latest revision as of 05:58, 21 October 2021

GSR, glutathione reductase

This DNA codes for GSR, also known as glutathione reductase, which catalyzes the conversion of glutathione to glutathione disulfide.

Usage and Biology

GSR is an enzyme that catalyzes the formation of glutathione. Glutathione is an antioxidant exists in humans, plants, fungi, animal that prevents cellular damage by reactive oxygen species. [1] In our project, we aim to deliver GSR to the lens, where GSH will be produced and prevent reactive oxygen species from damaging the lens proteins. It has been reported that age-induced cataract is mainly caused by the oxidation of the lens protein.[2] The delivery of GSR to the lens should help prevent cataracts from happening.

[1] Sius, H. (n.d.). Glutathione and its role in cellular functions.

[2] Spector, A. (n.d.). Oxidative stress-induced cataract: Mechanism of action.

Sequence and Features

Gel Pictures

We designed primers to remove stop codon and to move GSR cDNA to iGEM BioBrick. PCR was set up and the gel picture is shown below. Lane 1 is 1kb ladder. The expected size for GSR PCR check is ~1.8kb, which is shown in lane 5-8 (boxed in red).

Sequencing

We sent GSR out to sequencing after moving it to iGEM BioBrick. The sequencing results are unable to confirm the sequence from 480 to 744 bp. The four cutting sites are highlighted in yellow and GSR is highlighted in orange.

Sequence with vf2 primer:

Sequence with vr primer:

This GSR is later used in BBa_K1898250, which was also sent to sequencing. From the sequencing file snipped in part.igem.org/Part:BBa_K1898250, the GSR sequence from 480 to 744 bps is confirmed to be correct.

Contribution: LZU-HS-CHINA 2021

Characterization of Sortase BBa_K1898200 (and comparison to BBa_K3986001)

We found that GSR, glutathione reductase BBa_K1898200 may be able to reduce selenite to nano-selenium.

At the same time, we also found that two other enzymes may reduce selenite to nano-selenium. They are BBa_K3986001 and BBa_K3986002.

In order to facilitate the comparison and display of these three selenite reductases, we conducted a comparative experiment and summarized the experimental results as follows.

Functional verification and reduction rate comparison of three selenite-reduced proteins

The component correspondence is

BBa_K3986001----SAV 0956

BBa_K3986002----SAKG03 26900

BBa_K1898200----SAKOR 01018

In this study, the whole genome of LZ-01 was compared with the previously reported selenite reductase in the NCBI database, and three proteins with a similarity of 28-32% were obtained. The coding genes are SAV 0956, SAKG03 26900, and SAKOR 01018, respectively. See Table 2-1. Three genes were obtained by PCR from LZ-01, as shown in Figure 2-2. After that, the three genes were constructed on the pET 28a(+) inducible vector by the method of molecular cloning, and they were verified in vitro after purification by a nickel column. The verification results can be seen from Figure 2-3a, SAV 0956 gene expression has the strongest protein reduction ability, which reduced 59.73% Se (IV) after 0.5 h of reaction, followed by SAKOR 01018 which reduced 29.2% Se (IV). ), and SAKG03 26900 has almost no reduction ability. Therefore, we took the SAV0956 gene as the research object, and named the selenite-reduced protein expressed by it as SerV01. As shown in Figure 2-3b, the reaction centrifuge tubes from left to right are: control (no protein), the proteins expressed by SAKG03 26900, SAKOR, 01018SAV 0956, it can be observed that the control group has no red material at all, and the reduction gives the most red material Is the protein expressed by the SAV0956 gene.

Table 2-1 Results of gene comparison

Figure 2-2 PCR results of the three genes.

Fig2-3 The Se (IV) reduction rate of SAV0956, SAKG0326900, SAKQR01018with 1 mM Se (IV) in Tris-HCl 7.4.

The previously reported selenite reductase to verify the reducing ability has been identified, glutathione reductase in Escherichia coli, dehydrogenase I in Clostridium, and nitrite reduction in Serratia Enzymes, the dissimilation enzyme Srr in Bacillus selenide, and the NADH:flavin oxidoreductase of rhizobia are thought to be related to the reduction of Se(IV) in aerobic bacteria. The Serratia strain HCNT1 mutant lacking nitrite reductase grows poorly in the presence of 5 mM selenite, and fails to grow in the presence of 25 or 50 mM selenite, increasing selenite sensitivity . The selenite reductase CsrF in Alishewanella sp. WH16-1, the mutant constructed by the suicide allelic recombination method, showed the slow reduction of Se(IV). In Shewanella oneidensis MR-1, the reductase FccA was mutated to reduce selenite by 60%. Most studies have done knockout verification in bacteria, but there are few in vitro verification experiments after purification. Compared with other selenite reductases that have been reported, the enzyme used in this experiment was directly purified and verified in vitro. SerV01 enzyme alone can reduce selenite without the need for multiple enzymes, which is cost-saving and stable. With high performance, it is necessary to explore the applicability of its enzyme activity and reaction conditions.

SerV01 protein Se (IV) reduction enzyme activity dynamics analysis of Michaelis equation and determination of cofactor FMN

Exploring the reaction conditions of the enzyme, it can be seen from Figure 2-4 that when NADH is added as an electron donor, there is a significant decrease in absorbance value from 0 to 150s under the real-time wavelength detection system of U-3900H instrument, indicating the reduction of selenite Enzymes can be combined with electron donors to achieve a reduction effect. There was no change in absorbance in the group without added protein, indicating that NADH was not consumed.

Figure 2-4 Changes in absorbance values for whether NADH is bound or not.

SerV01 protein is similar to the old yellow enzyme family, we further study its enzyme activity kinetics in order to explore the enzyme activity. It can be seen from Figure 2-5 that the Vmax value is 1.119 mol NADH mol-1 s-1 and the Km value is 15.5 μmol/L. Compared with other reductases, the in vitro Se(IV) reduction Vmax of TrxR enzyme in Bacillus Y3 is 12.23 μmol/L, 11.20 μmM min-1 mg-1. For the Srr in Bacillus mLS10, the Km of the enzyme is 145 ± 53 μmol/L, the Vmax is 23 ± 2.5 μM min -1, and the Kcat is 23 ± 2.68 s -1. Enzyme kinetics showed that the low Km value of the enzyme indicates that the enzyme has a high binding capacity to the substrate. Therefore, the selenite reductase SerV01 can be regarded as an effective enzyme for the production of SeNPs.

Figure 2-5 Enzyme activity kinetic curves.

Due to previous reports of NADH flavin oxidoreductase in Rhizobium selenitireducens, dehydrogenase I (Hydrogenase I), sulfite reductase and glutathione peroxidase in Clostridium pasteurianum are all sub- Selenate reductase is used for comparison with the sequence of the target protein SerV01. The Mega 7.0 software was further used to compare SerV01 with protein products of different genera. The results showed that this protein belongs to NADH-dependent flavin redox protein, which is similar to rhizobia selenitireducens NADH: flavin oxidoreductase, and is similar to the known flavin oxidoreductase. Traditional sulfite reductase is similar to selenite reductase from different sources, as shown in Figure 2-6.

Figure 2-6 SerV01 system evolutionary tree

The SerV01 protein structure predicted by the 3DLigandSite server is shown in Figure 2-7. The gray part is the protein backbone, the blue part is the predicted ligand binding site, and the green part is the predicted ligand FMN. As shown in the figure, the ligand is in The protein binds in the pocket and transfers electrons through NAD(P)H to achieve the function of reducing tetravalent selenium to zero valent selenium.

Figure 2-7 Protein structure and ligand mockups.

As the ligand is speculated to be FMN, high performance liquid chromatography was used for verification. The FMN standard sample was prepared as a control, and the prepared ligand solution of SerV01 protein was used as the experimental group to obtain the corresponding absorption peaks, as shown in Figure 2. -8. After measurement, it was found that the peak was separated at 3.05 at the same time, thus proving that the ligand was indeed FMN. The previously reported CsrF selenium-chromium co-reductase also uses FMN as a ligand. The ChrT gene encodes a chromate reductase, also known as flavin mononucleotide (FMN) reductase, which can catalyze the reduction of Cr (VI). ChrR in is also a hexavalent chromium reductase with FMN as a ligand. FMN has become a common ligand for redox proteins in bacteria because the partial dehydrogenation and hydrogenation reaction of isooxazine is a reversible process that participates in the redox reaction of enzymes.

Figure 2-8 Peak time graph of ligands determined by HPLC