Step 1 Learning
From previous studies and our understanding based on fundamental researches of papers, there are some Selenium-reduction bacteria existing, which can be categorized as three groups, Intracellular reduction, extracellular reduction and inter-extra simultaneous presence. The first category includes the Desulfovibrio desulfuricans, Escherichia coli, Ralstonia metalliduransVeillonella atypica, Wolinella succinogenes, The Pseudomonas fluorescens; class II includes the Geobacter sulfurreducens, Thauera selenatis, Enterobacter cloacae, Shewanella oneidensis; The third category is the most, which includes the Rhodospirillum rubrum, Bacillus cereus, Selenihalanaerobacter shriftii, Bacillus selenitireducen, and etc.
However, these bacteria have difference in their efficacy of reducing the toxicity of selenite salt. There were evidence in those studies that revealed there were still plenty of flaws in this research, such as the deficiency of reduction rate of those bacteria in high solvent concentration caused by the low tolerance for the Selenium. Huang Shengwei and his team concluded that the maximum selenium tolerance concentration was ≤ 100mM. It indicates that a novel and advanced strain should be selected in the future to promote the SeNPs production.
Step 2 Designing
Therefore, what we planned to do is to do the engineering and construction of a novel Selenium reduction bacteria with higher tolerance and efficacy, by applying the concept of synthetic biology. We have to select a efficient Selenium reduction strain and select its Selenium reduction gene by conducting similarity comparison in NCBI database. Furthermore, we decided to implement the microbial cell surface display system, enhancing the stability and reaction efficiency of reagent enzymes, we also prevents the degradation of foreign proteins by intracellular enzymes produced by the host, eliminating the disadvantages such as the inability of certain proteins or peptides to undergo specific folding within the cell, as well as eliminating the additional cost of enzyme purification and immobilization. In this system, we select ice nucleation protein(INP) as our carrier protein, since it has the ability of stably expressing exogenous large weight protein. We also decided to apply Nissle1917(EcN) as our chassis, since it is a widely used probiotic that is permitted to be used in human bodies by institutions like FDA. To eliminate its cryptic plasmid, we decided to implement pSB1A3, a high copy plasmid, to stably carry our fusion protein.
P1. The framework of the implementation of INP
P2. The plasmid profile of pSB1A3.
P3. Framework of design.
Step 3 Building
To achieve the goal, we first do the selection of powerful Selenium reduction bacteria to find its functional gene and the reuductase it encodes. We successfully designed and registered new parts in this process.
Figure 1. The frame of the coding sequence of SAV 0956, the other two used different appropriate primers instead of SAV-F.
Comparison of the whole LZ-01 gene with the other already reported selenite reductase in the NCBI database yielded three potentially reductive genes, respectively SAV 0956, SAKG03 26900, SAKOR 01018. To validate the function of the three genes in Se (IV) reduction, three pairs of primers were designed to amplify the genes from LZ-01. After the fragments were digested with EcoRI and XhoI, the proteins were inserted into the plasmid pET-28a (+) by a ligation reaction and the 6 × His tag was designed.The nickel column hangs the target protein carrying 6 × His, and further elution can purify the product of the target protein.
Figure 2. The results of gene comparison
Figure 3. The Primers we used and their sequence in the experiments.
Figure 4. The PCR results of the three genes
Among the three expressed proteins, SerV01 derived from SAV0956 has the highest reduction ability and was selected by us to do the further study. Phylogenetic comparison of SerV01 with protein products from different genera using Mega 7.0 software showed that this protein belongs to the NADH-dependent flavin redox protein, similar to the rhizobium selenitireducens NADH: flavin oxidoreductase. The SerV01 protein structure predicted by the DLigandSite server is shown in Figure 5 where the gray part is the protein skeleton, the blue part is the predicted ligand binding site and the green part is the predicted ligand FMN, shown in the ligand binding in the pocket of the protein, delivering electrons via NAD (P) H to achieve the function of reducing tetravalent to zero valent selenium.
Figure 5. SerV01 system evolutionary tree
In our next process, the SerV01 protein derived from LZ-01 was successfully anchored to the extracellular membrane of the probiotics EcN using the cell surface display technology. Gene sequences of the carrier protein INP, strong promoter and the expression vector were obtained, respectively, and primers were designed to fuse the INP to the target protein using Overlap PCR amplification technology.The fusion proteins were combined to the expression vector pSB1A3 using homologous recombination.
Figure 5. The frame of the combination of INP-N and SerV01
Figure 6. The frame of the design of the fusion protein and the plasmid (pSB1A3-INP-SerV01).
The process of construction of fusion protein and the plasmid vector are listed as follows:
(1) Selection of carrying proteins.The N-terminal domain of the ice crystal nuclear protein (INP-N) stored from this laboratory was used as a template to design the primer INP-F, INP-R. using Snapgene software
(2) Selection of passenger protein. The SerV01 sequence was selected as the passenger protein, the primers were designed to copy the gene sequence down from the LZ-01, and the forward-reverse primer serv-F, serv-R. was designed
for Overlap PCR Strong promoter and gap sequences were added to the front end of the serv-f primer to allow for more subsequent expression of the fusion protein.
(3) To eliminate the multiple secret plasmids that may have effects on subsequent live enzyme expression, so high copy plasmid was required for this study, and constituent promoter Plac plasmid pSB1A3 was found to be successfully
transformed into EcN.
(4) Cloning and transformation of the engineering strains. The INP-SerV01 was linked together after Overlap PCR amplification to connect the fusion gene fragment for recombination purposes and the plasmid pSB1A3 by homologous recombination.
Blunting Kination Ligation (BKL) Kit first linearized its circular DNA molecules and subsequently incubated with the target fragment.Recombinant plasmid containing the INP-SerV01 fusion polypeptide was named pSB1A3-INP-SerV01.
Step 4 Testing
The engineered fusion protein expressed on the chassis in vitro successfully expressed higher reduction efficacy as shown in the figure:
