The main goal of our work is to develop a functionalized cellulose-based biomaterial for bone and cartilage regeneration in order to allow a faster injuries recovery time and overcome remaining challenges in repairing these connective tissues. In biomedicine, emergent strategies have focused the use biomaterials loaded with drugs.^1-3 We aimed to use bacterial cellulose, a biocompatible biomaterial, as scaffold for the delivery of the human bone morphogenetic protein 2 (BMP2). It is worth to note that the diffusion of a drug far of the action site can become ectopic tissue formation, representing a serious problem. Therefore, we focused to attach the BMP2 by the use of a cellulose binding domain (CBD). We choose the CBD of the scaffolding protein of C. thermocellum (CBD cipA) because the higher affinity compared to other CBDs, reported by the Team Imperial_2014. Moreover, in an effort for a longer broad application of a functionalized bacterial cellulose, such as wound healing, we also fused the CBD cipA with an elastin like polypeptide (ELP_C5) derived from the ELP- [V-150].

BACTERIAL CELLULOSE

Figure 1. Surface topography imaged by AFM corresponding to a sample of dried bacterial cellulose.

High levels of cellulose is the first module of our project. Thus, we have focused to cotransform two plasmids in E. coli BL21 (DE3) because it is resistant to toxic proteins. The firs plasmid is the operon bscABCD for cellulose biosynthesis while the second one is conformed by the genes Cmcax and CcpAx which are members of the cellulose synthase complex and allow an enhanced cellulose production rates.^4,5 Because the difficulties of chemical synthesis in four of the six genes, it was necessary to add aptamers at 5’ ands 3’ and the Gibson assembly, using psb1c3 as the host vector was delayed. To date, we have successfully assembled the plasmid containing the genes Cmcax and CcpAx.

Furthermore, we started the atomic force microscopy (AFM) analysis of the bacterial cellulose, analyzing its topography as the first step to approximate the total cellulose binding sites where the fusion protein will bind. This analysis will allow us to know the drug concentration in the cellulose.⁶

FUSION PROTEINS

CBD cipA-BMP2

Figure 2. Tridimensional structure of CBD cipA-BMP2 obtained from the server I-Tasser.

Two gBlocks were synthetized for in-frame protein assembly. The first gBlock consisting of lacI promoter, RBS and CDS of CBD cipA while the second gBlock containing the CDS of BMP2. Both of them were codon optimized for E. coli.

Because it has been demonstrated that the C-terminal domain of BMP2 provides osteogenic activity, ⁷ we intended to leave it free. Thus, it was necessary to fused it C-terminally to an N-terminal CBD.⁸ We decided to choose the CBD cipA because the higher affinity to bacterial cellulose compared to other CBDs that was reported by the team Imperial 2014.⁹

The CBD cipA has originally two linkers. The N-terminal linker is a signal sequence typical of the bacterial extracellular proteins while the C-terminal linker separating the CBD cipA from Coh3 module.

Then, in contrast to the team Imperial 2014, we decided to use only the C-terminal linker hypothesizing that this can provide a better binding strength than the use of two linkers.

Figure 3. Schematic overview of the CBD cipA-BMP2 construct cloned in psb1c3.

CBD cipA-sfGFP

Figure 2. Tridimensional structure of CBD cipA-sfGFP obtained from the server I-Tasser.

The gBlocks synthetized for in-frame protein assembly corresponded to the first gBlock consisting of lacI promoter, RBS and CDS of CBD cipA; the second gBlock containing the CDS of sfGFP with a 6X His tag. Both of them were codon optimized for E. coli.

We aimed to add a 6X His tag at C-terminus in order to visualize and measure at single-molecule level the interaction between the CBD cipA and bacterial cellulose.⁶

Figure 5. Schematic overview of the CBD cipA-BMP2 construct cloned in psb1c3.

CBD-ELP_C5

Figure 6. Tridimensional structure of CBD cipA-ELP_C5 obtained from the server I-Tasser.

ELP_C5 was obtained via overhang PCR using primers containing prefix and suffix in RFC25, and the plasmid pET-24a-ELP[V-150] as template.

Each ELP undergoes a Tt that leads to its aggregation or solubilization. The Tt of an ELP fusion protein is different to that of the free ELP depending of the surface accessible surface. Because we aimed a future clinical application of ELP fusion proteins in aggregated state, we decided to use ELP[V-150] with a Tt=28.2℃,¹⁰ lower than the corporal temperature. However we have cloning a 450 bp fragment of ELP[V-150] and we expect a similar Tt.

Figure 7. Schematic overview of the CBD cipA-ELP_C5 construct cloned in psb1c3.

CHARACTERISTICS OF C-lastin

In an effort to overcome the unsolved challenges in bone healing that are mainly derived from pathological and age related events, C-lastin takes advantage of the biocompatibility of bacterial cellulose to develop a functionalized biomaterial for bone and cartilage regeneration.
The principle of C-lastin is a post-hoc functionalization of a chimeric protein formed by a CBD and the human BMP2 with bacterial cellulose functionating as scaffold.
The fusion protein containing the ELP_C5 attached to bacterial cellulose via CBD cipA can confer elastic properties to the biomaterial tapping it for biomedical applications such as wound healing.

REFERENCES

1. Mohtaram, N.K., Montgomery, A., Willerth, S.M. (2013).Biomaterial-based drug delivery systems for the controlled release of neurotrophic factors. Biomedical biomaterials, 8(2):022001
2. Shi, Q., Li, Y., Sun, J., Zhang, H., Chen, L., Chen, B., Yang, H., Wang, Z. (2012).The osteogenesis of bacterial cellulose scaffold loaded with bone morphogenetic protein-2. Biomaterials, 33(28):6644-9
3. Winkler, T., Sass, F. A., Duda, G. N., & Schmidt-Bleek, K. (2018). A review of biomaterials in bone defect healing, remaining shortcomings and future opportunities for bone tissue engineering: The unsolved challenge. Bone & Joint Research, 7(3), 232–243. http://doi.org/10.1302/2046-3758.73.BJR-2017-0270.R1
4. Römling, U., & Galperin, M. Y. (2015). Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions. Trends in Microbiology, 23(9), 545–557. http://doi.org/10.1016/j.tim.2015.05.005
5. Buldum, G., Bismarck, A., & Mantalaris, A. (2018). Recombinant biosynthesis of bacterial cellulose in genetically modified Escherichia coli. Bioprocess and Biosystems Engineering, 41(2), 265–279.
6. Zhang, M., Sheng-Cheng, W., Zhou, W. & Xu, B. (2012).Imaging and Measuring Single-Molecule Interaction between a Carbohydrate-Binding Module and Natural Plant Cell Wall Cellulose. J. Phys. Chem. B, 2012, 116 (33), pp 9949–9956
7. Schmoekel, H. G., Weber, F. E., Schense, J. C., Grätz, K. W., Schawalder , P., & Hubbell , J. A. (2005). Bone repair with a form of BMP-2 engineered for incorporation into fibrin cell ingrowth matrices. Biotechnology Bioengineering, 89(3):253-62.
8. Yaniv, O., Morag, E., Borovok, I., Bayer, E. A., Lamed, R., Frolow, F., & Shimon, L. J. W. (2013). Structure of a family 3a carbohydrate-binding module from the cellulosomal scaffoldin CipA of Clostridium thermocellum with flanking linkers: implications for cellulosome structure. Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 69(Pt 7), 733–737.
9. Team Imperial College London. (2014). Aqualose. Recovery from https://2014.igem.org/Team:Imperial
10. Tang, N. & Chilkoti, A. (2016).Combinatorial codon scrambling enables scalable gene synthesis and amplification of repetitive proteins. Nature Materials, 15, pages 419–424

Team:Ecuador/Judging

DESIGN

Index