Team:Ecuador/Background
PROJECT BACKGROUND
iGEM
TEAM
ECUADOR
PHASE 1 BACTERIAL CELLULOSE
Bacterial cellulose (BC) is a glucose polysaccharide that exhibits numerous properties such as unique nanostructure, high water holding capacity, high degree of polymerization, mechanical strength and high crystallinity
These properties clearly show that BC has tremendous potential and provide a promising future in various fie.
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PHASE 2 FUSION PROTEIN CBD-ELP-BMP2
This fusion protein is composed by the cellulose binding domain cipA (CBD cipA) that exhibits a high affinity to cellulose, followed by an elastin like polypeptide (ELP) which allows us an easy and rapid purification and finally the bone morphogenetic protein 2 (BMP-2) that is a potent osteoinductive cytokine capable of inducing bone and cartilage formation.
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PHASE 3 BIOMATERIAL FUNCTIONALIZATION
In order to get an elastic and functional biomaterial the fusion protein CBD-ELP-BMP2 was coupled to bacterial cellulose by a crosslinking process. This biomaterial can be used in biomedical applications focused on bone and cartilage formation because of its compatibility with the human body.
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Phase 1: BACTERIAL CELLULOSE
Cellulose was the most common biopolymer in the world. The primary form in which the material is found is lignocellulotic in trees, however there are other sources such as bacterial cellulose [1].This was first described by Luis Pasteur in the previous century and reported for the first time its use in a Philippine dessert called coconut cream, however, it was not until 1886 when it was reported as a type of cellulose in an acetic fermentation, after being observed as a floating film in a culture medium[2]. In recent years, several studies have been carried out on the usefulness of bacterial cellulose due to its high degree of purity and its simpler structure than that obtained from plants, in addition to the speed of polymer formation, reducing costs and environmental impact in the purification process to eliminate the lignin and other impurities of the material to be applied in the industries[3].
Bacterial cellulose has been used mainly in the paper industry, in the food for the realization of various desserts and strong dishes and as a material for garment development, due to its great flexibility, it is also impregnated with several nanoparticles to give antimicrobial, antifungal capacities[4]. Its ability to be combined with other proteins gives it the advantage to create new polymers with other desired properties such as bioplastics and drug administrators when combined with therapeutic proteins[5]. The existing biocompatibility between bacterial cellulose and human cells has led to the use of the polymer as a matrix for the regeneration of organs and tissues such as cartilage and skin[6].
References
1. Ummatyotin, S., & Manuspiya, H. (2014). A critical review on cellulose: From fundamental to an approach on sensor technology . Renewable and Sustainable Energy Reviews, 402-409.
2. Iguchi, M., Yamanaka, S., & Budhiono, A. (2000). Review bacterial cellulose-a masterpiece of nature's art . Journal of material science, 261-270.
3. Foresti, L., Vazquez, A., & Boury, B. (2016). Appiation of bacterial cellulose as precusor of carbon and composites with metal oxide, metal sulfide and metal nanoparticles. Carbohydrate polymers.
4. MAneerung, T., Tokura, S., Rujiracanit, & R. (2007). Impregnation of silver nanoparticles into bacterial cellulose for antimicrobial wound dressing. Carbohydrate polymers, 43-51.
5. Helenius, C., Backhdal, H., Bodin, A., Nannmark, U., Gatenholm, P., Risberg, & B. (2005). In vivo biocompatibility of bacterial cellulose. Wiley InterScience, 431-438.
6. Backdahl, H., Helenius, G., Bodin, A., Naanmmark, U., Johansson, R., Risberg, B., & Gatenholm, P. (2006). Mechanical properties of bacterial cellulose and interactions with smooth muscle cells. Biomaterials, 2141-2149.
About our project
Phase 2: FUSION PROTEIN CBD-ELP-BMP2
LASTIN-LIKE POLYPEPTIDES
Elastin-like polypeptides (ELP) are genetically encodable artificial biopolymers. They are elastomeric proteins formed by a repetitive pentapeptide of Val-Pro-Gly-Xaa-Gly sequence, Xaa can be any amino acid except proline. [1].
ELPs are thermostable biopolymers whose properties vary depending on the temperature, pH or ionic strength. They can pass from a soluble state to an insoluble one and reversibly depending on their transition temperature (Tt) [2], at temperatures lower than the Tt ELPs are soluble, but insoluble when the temperature exceeds the Tt. This property is maintained even when they are fused with other proteins and has been used in protein purification. The amino acid residues that contain groups susceptible to ionization result in a polymer with a Tt regulated by changes in pH, in addition, the substitution of the Xaa residue allows ELP to be designed with a desired Tt[3].
In biomedicine, ELPs have applications in the specific drug delivery, in tissue engineering and regenerative medicine. It has been possible to selectively transport antineoplastic drugs to pathologically changed tissues, allowing the polymer-drug conjugates to accumulate in the vicinity of a tumour, showing a lower toxicity compared to free-running drugs. [1].
In regenerative medicine, ELPs have been used as scaffolds in tissue regeneration, and have shown promising results in treatments for articular cartilage damage, where a hydrogel made of ELP is used, in which it effectively contributed to the production of a cartilage matrix. Other studies show that ELPs conjugated with polymers such as polyacrylic acid and polyethyleneimine can strongly influence the aggregation, morphology and differentiated function of hepatocytes in vitro, showing the ability to use ELP in the regeneration of liver tissue [1]. In addition, ELPs have shown promising results to be used in the engineering of ocular surface tissues, and in vascular grafts [4].
References
1. KOWALCZYK, Tomasz, et al. Elastin-like polypeptides as a promising family of genetically-engineered protein based polymers. World Journal of Microbiology and Biotechnology, 2014, vol. 30, no 8, p. 2141-2152.
2. PARK, Ji-Eun; WON, Jong-In. Thermal behaviors of elastin-like polypeptides (ELPs) according to their physical properties and environmental conditions. Biotechnology and Bioprocess Engineering, 2009, vol. 14, no 5, p. 662.
3. MCMILLAN, R. Andrew; CONTICELLO, Vincent P. Synthesis and characterization of elastin-mimetic protein gels derived from a well-defined polypeptide precursor. Macromolecules, 2000, vol. 33, no 13, p. 4809-4821.
4. MARTÍNEZ-OSORIO, Hernán, et al. Genetically engineered elastin-like polymer as a substratum to culture cells from the ocular surface. Current eye research, 2009, vol. 34, no 1, p. 48-56.
In this project a new biomaterial based on the cross-linking of bacterial cellulose (bacterial celluose, BC) and elastin-like polypeptides (Elastin like polypeptides, ELP) will be developed, for use in biomedical applications. The cross-linking of both materials will be carried out with synthetic biology techniques using the Escherichia coli bacterium as the expression system of the new biomaterial, and as a mediator of the cross-linking to two protein modules of the carbohydrate binding domain (carbohydrate binding domain, CBD) specific for cellulose and one module of bone morphogenic protein 2 (BMP-2).
Bacterial cellulose will be used as a bandage matrix, due its biocompatibility in vivo with the body, moreover its ability to provide an optimal three-dimensional substrate for cell attachment and a microfibrillar structure that provides flexibility, and its high retention capacity of water and gas exchange, which allow it to obtain rapid rates of epithelization and tissue regeneration. CBDs are modules of proteins found in carbohydrate enzymes whose function is to bind to cellulose carbohydrate, allowing the ELP protein to give greater flexibility to the bandage, while the BMP2 protein, which is an inducer of cell differentiation in osteoblasts, will be responsible for reducing the recovery time of the bones.
To achieve the objective, the expression of the cellulose and the fusion protein in two bacteria will be carried out separately. For the synthesis of bacterial cellulose, an E. coli expression system will be used; introducing two plasmids: psb1C3 responsible for cellulose synthesis and psb1A3 responsible for the synthesis of the export system and overproduction. For the production of the fusion protein, plasmid psb1C3 will be used, which will contain the genes for the proteins CBD, ELP and BMP2
Bacterial cellulose will be used as a bandage matrix, due its biocompatibility in vivo with the body, moreover its ability to provide an optimal three-dimensional substrate for cell attachment and a microfibrillar structure that provides flexibility, and its high retention capacity of water and gas exchange, which allow it to obtain rapid rates of epithelization and tissue regeneration. CBDs are modules of proteins found in carbohydrate enzymes whose function is to bind to cellulose carbohydrate, allowing the ELP protein to give greater flexibility to the bandage, while the BMP2 protein, which is an inducer of cell differentiation in osteoblasts, will be responsible for reducing the recovery time of the bones.
To achieve the objective, the expression of the cellulose and the fusion protein in two bacteria will be carried out separately. For the synthesis of bacterial cellulose, an E. coli expression system will be used; introducing two plasmids: psb1C3 responsible for cellulose synthesis and psb1A3 responsible for the synthesis of the export system and overproduction. For the production of the fusion protein, plasmid psb1C3 will be used, which will contain the genes for the proteins CBD, ELP and BMP2
Phase 3