Corresponding author: João Vitor Dutra Molino (
Academic editor:
In this project, we propose to explore the modular characteristic of spider silk proteins, through synthetic biology techniques, by combining and directing its properties to the desired application. The aim of this project is to generate a modular bionanomaterial able to immobilize proteins. This bionanomaterial will be composed of modular recombinant proteins from spider silk, which will be the immobilization support to other proteins, in this project an antimicrobial protein (enzybiotic). By combining these proteins and their properties, the primary focus will be the use of this technology for the development of artificial skin for burn victims.
The recombinant proteins, spider silk proteins and enzybiotics, will be expressed in
Immobilization techniques are applied to a wide range of treatments and processes, from medical applications to biotransformations in industrial plants. The industrial/commercial application of biomolecules such as proteins depends on the stability and functionality of the process employed. This process often differs from the natural environment of proteins in terms such as temperature, presence of organic solvents and pH values. Consequently, techniques such as immobilization can promote stabilization and add functionality even when these biomolecules are under different environmental conditions. This stabilization is normally achieved by protein binding to a scaffold (
The aim of this project is to use this biomaterial for protein immobilization. Initially, we will immobilize enzybiotics for application to burn wounds, as a model to test this support. However, there are other possible applications with economic and academic interest. Fig.
The retention of molecules inside a reactor, or an analytical system, is described as immobilization, whose purpose is to improve protein stability, selectivity, and particularly for the enzymes, increase catalytic activity (
Enzymes are widely used in our society, and their applications range from industrial processes - such as in food production, biofuels and tissue - to more complex therapeutic applications, such as biopharmaceuticals (e.g., asparaginase use for acute lymphoblastic leukemia treatment (
A medical application that benefits from enzyme immobilization is biosensor development (
Enzybiotics comprise a class of enzymes with antibiotic activity. These enzymes are able to fight resistant bacteria, such as MRSA, VRSA and VISA (
The polymer constituting the spider silk bears interesting properties for various applications, including immobilization of molecules such as proteins. Spider silk is known mainly for its tensile strength and fracture resistance, but also exhibits elasticity, adhesion, biocompatibility and low degradation. Its strength can be compared to Kevlar synthetic polymer, which is composed of aramid and is used in for manufacturing body armor (
It is known that certain repetitive sequences of amino acids confer specific properties to these structures and proteins in tissue, allowing one to obtain materials with desired characteristics through genetic manipulation of these structural domains. The poly-alanine domains (poly(A/GA) (Glycine-Alanine) in MaSp1 proteins, MaSp2 and MISP are associated with formation of beta-sheets and the production of strong fibers, while repeating sequences "GPGGx" and "GGX" as in Flag protein, preferably generates an elastic beta-spiral region, which provides elasticity (
In addition, terminal domains (N-terminal NT and C-terminal CT) are highly conserved both among species and different types of silk (
There is some evidence of dependence on N- and C-terminal domains for polymerization, which can lead to interesting technological possibilities. The adaptation of these domains flanking a protein of interest opens the possibility of its immobilization if spun alongside “native” spider silk proteins. Moreover, core structural domains’ ("poly(A/GA" and "GPGGx" and "GGX") customization influence the physical properties of the silk (
Heterologous proteins are produced in several established expression systems, such as
Therapeutic proteins are preferably produced in transgenic mammalian cell systems, because of their ability to express and correctly fold proteins. However, its production cost is high, especially when compared to plants as expression systems. Molecules such as monoclonal antibodies (mAbs) are mainly produced in mammalian cells and their average production cost in this system is estimated to be $ 150.00 per gram of raw materials, whereas production in plant systems costs approximately US$0.05 per gram (
However, the studies on genetic engineering using microalgae are incipient and present challenges. The main challenge is the low productivity of recombinant proteins expressed in the nuclear genome, hindering commercial applications to date (
Evaluate production capacity of synthetic spider silk proteins (based on MaSp1 and MaSp2) and protein chimeras with NT and CT domains flanking enzybiotics in
Set methods for polymerization of silk produced by microalgae;
Test biopolymer and antibiotic properties of spun spider silk, pure and in combination with enzybiotic chimeric proteins;
Assess the project development in an iGEM competition context regarding its scientific achievement and the real-time openness in all process steps: idealization, laboratory procedures, results and discussion
Many obstacles need to be overcome for the effective production of a biomaterial such as a recombinant spider silk capable of immobilizing proteins in its matrix. With this purpose, this project offers solutions, as of yet untested, for example: the use of microalgae as a production platform for the expression of spider silk proteins, as well as chimeras with NT and CT domains, and flanking enzybiotics. Research on transgenic microalgae are driven by the global demand for recombinant proteins and other bioproducts. This biotechnological market is growing exponentially, it has reached 140 billion dollars in sales as of 2013, and it continues to grow (
More than developing a product that could help thousands of patients, the development of an antibiotic chimeric biopolymer in
It is important to highlight the fact that this project will be carried out at an international competition dedicated to the development of high-level research in Synthetic Biology (iGEM - International Genetically Engineered Machine) with an open and integrative approach. This competition takes place annually in Boston, USA, and it stimulates interdisciplinary groups to problem solving through genetically modified organisms. In line with this proposal, the team responsible for this project consists of undergraduate and graduate students from various institutes of the University of São Paulo. The blending of open approaches and such interdisciplinary groups contributes to the development of the research
The vector pBluescript II (Thermo Fisher Scientific Inc.) will be used. The constructed cassette will be flanked by the restriction sites
The native proteins MaSp1 and MaSp2 (
The
Recovered cells will be plated on TAP agar medium with increasing antibiotic concentrations (0.1, 2 and 5 μg/mL Zeocin). Candidate transformed colonies, displaying high Zeocin resistance, will be analyzed through PCR screening, and PCR positives colonies will be tested for protein of interest production by Western blot. Basically, the mutant cells are cultured as described above and fractions of the supernatant and cell lysate will be tested for the presence of the protein of interest. Cell lysis will be accomplished by sonication as described in the literature (
Samples of supernatant and total soluble proteins will be denatured by adding SDS-PAGE loading buffer (Laemmli) with β-mercaptoethanol, followed by incubation at 95 °C for 5 min. Proteins will be separated on 12% polyacrylamide gels at 120-150 V and transferred to nitrocellulose membrane at 200mA for 1h. Then they will be blocked in 5% solution of skimmed milk and the protein of interest will be probed with monoclonal mouse anti-His antibody. The membrane will then be washed 3 times with TBS-T (Tris-buffered saline with Tween 20 detergent) for 10 min and incubated with anti-mouse antibody conjugated with alkaline phosphatase, 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitroblue tetrazolium (NBT) substrates which forms an insoluble dark blue diformazan precipitate, allowing protein identification.
Purification of the proteins of interest will be carried out by Nickel resin (Ni Superflow His60, Clontech®) following the manufacturer's instructions. The method is based on the affinity interaction with the hexa histidine tail, present in recombinant protein with the already mentioned resin. Basically, the sample is added to the column with the resin precharged with nickel ions, in which the proteins of interest containing histidine residues on its surface will be attached. Proteins not bound to the column will be washed out with the wash buffer, while the protein of interest is eluted with buffer containing 500 mM Imidazole.
Quantification of purified proteins will be obtained via the Enzyme Linked Immunosorbent Assay (ELISA). Thus 200 μL of sample are incubated in 96-well plates at 37 °C for 30 min, then the solutions are removed, blocked in 5% solution of skimmed milk and the wells are washed 3 times with TBS-T (Tris-buffered saline with Tween 20 detergent) . Then, 200 μL/well of TBS-T solution of monoclonal mouse anti-His is added and incubated at room temperature for 2 h and washed as described above. A new TBS-T solution with anti-mouse monoclonal antibody conjugated with alkaline phosphatase is added, incubated at room temperature for 2 h and washed 3 times with TBS-T for 10 min. For the development, a freshly prepared solution of p-nitrophenyl phosphate is added and incubated in the wells for 30 min, and the plate is subsequently read in a plate reader at 405 nm.
Results will be evaluated by analysis of variation (ANOVA) performed in Statistica software 10. Statistical significance will be evaluated by estimating the descriptive level (p) and the results will be considered statistically significant at p < 0.05 (confidence level of 95%) .The methods described above are shown in the flowchart in Fig.
We would like to thank Nicolau de Almeida for reviewing the manuscript for the use of English.
Project overview. Schematic representation of spider web structure from macro to nano scale. A representation of: enzybiotic protein from a bacteriophage; a spider silk protein with repetitive domains and N and C terminals; host expression system
Schematic representation of spider silk proteins and chimeric protein.
Cassette construction to be inserted in
Experimental Flowchart. (A) Wild Cells incubated with built vectors. (B) Wild-cell transformation by electroporation. (C) Selection of mutants resistant to Zeocin. (D) Screening of antibiotic resistant cells by PCR. (E) Cultivation of PCR positive cells. (F) Fractions to be tested for the presence of recombinant proteins. (G) Detection of recombinant proteins present in the fractions by Western Blot. (H) Protein Purification. (I) Quantification via ELISA. (J) Spider silk polymerization reaction.
Desired proteins to be expressed in
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MaSp1 |
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CDS fully sequenced |
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MaSp2 |
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CDS fully sequenced |
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NTD + MV-L + CTD | Bacteriophage phiMR11 | Effective against MRSA, VRSA e VISA |
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NTD + LysK + CTD | Bacteriophage K | Effective against MRSA e VRSA |
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NTD + Lysostaphin + CTD |
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Effective against MRSA, ORSA e VISA |
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Chronogram of execution
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Plasmid construction | X | ||||
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Transformed strains evaluation | X | X | X | X | |
Cob polymerization evaluation | X | X | |||
Results analysis | X | X | X | X | X |
Wiki development | X |