Team:USP UNIFESP-Brazil/Description
AlgAranha Team USP_UNIFESP-Brazil
Spider silk is an amazing bionanomaterial due to its exceptional features. It exhibits high tensile strength, high fracture resistance, high elasticity and it has biocompatibility and low degradability. Some studies show silk can be compared to a synthetic polymer called Kevlar, that is made of aramid and is used to make body armor (Lewis). Silks are polymers composed of proteins. These proteins and their genes present specific repetitive sequences that are associated with silk properties, making it possible to handle it in order to obtain the most suitable material for each application. Moreover, some studies suggest that the terminal domains of silk proteins play important roles in the polymerization process. In this way, it can be possible to add N- and C- terminal domains in several interesting proteins or motifs and spun it with native proteins to obtain a customized silk.
The size of genes, their repetitive sequences and high content of Cytosine and Guanine establish a challenge to many expression systems. However Chlamydomonas reinhardtii has naturally high CG content in its genome and it could be able to deal with this problem, it can also perform post translational modifications and it can secrete complex proteins, easing the purification process. Furthermore, it exhibits low cost production, rapid growth, scalability and stable transgenic line generation, all desired characteristics to an expression system (Wijffels, Rosenberg). It is important to highlight the low cost of microalgae cultivation, while production of antibodies in mammalian cells presents an average cost about US$150.00/per gram of raw materials and plants US$0.05/per gram (Dove), Maylfield has estimated that microalgae can reach US$ 0.002/liter. Chlamydomonas reinhardtii also is Generally Recognized As Safe (GRAS), that is, it has low risk of being contaminated by virus, prions and bacterial endotoxins (Mayfield). Easy purification and low risk of contamination are very significant issues when we are talking about making products to biomedical applications.
Figure 1. Cassete
Our project was inspired by the UCLA’s projects presented at iGEM in 2014 and 2015 about synthetic silks. Like we said above, silks are very interesting material but one of the main problems is dealing with its high content of Cytosine and Guanine. Most of the expression systems are not able to deal with that, however we have known Chlamydomonas reinhardtii has naturally high CG content which makes it interesting as an expression chassis. One of UCLA’s projects was called Silk Functionalization: Developing the Next Generation of Performance Fibers. The team has created a genetic construct using the Green Fluorescent Protein (GFP) inserted between N- and C- terminal domains of Bombyx mori’s silk to express proteins able to bind to native silk proteins. When this customized GFPs and native proteins were mixed and spun they were able to recognize each other by the terminal domains, making a fluorescent silk.
From this idea we thought: “Silk is a very interesting material and could be expressed in Chlamydomonas reinhardtii, a model organism that has many advantages and that remains underexplored. What could be put in place of a GFP to express in microalgae? What biomolecule could take advantage of this amazing silk feature? It has to work while immobilized... Why not enzymes? There are many studies about enzymes immobilization, enzymes are very appropriate and useful in many applications around the world!”. But we knew it could not to be any enzyme linked to silk, we knew it need to address technical, social and economic issues. So we read that spider silk has been studied to many biomedical applications, like the treatment of burn victims. Then we studied about burns in order to find an interesting enzyme that could solve some problem and we learned that one of the most important problems affecting burn victims is sepsis. In this way, we sought for antimicrobial enzymes and we found the endolysins. Endolysins are very interesting enzymes because they are able to combat multiresistant bacterias and they are specific, avoiding problems like the antibiotic resistance.
So we decided to make a project about spider silk production in microalgae, creating possibilities to future works with silk proteins from this amazing organism model, like the association between them and endolysins by UCLA’s approach in order to make an antimicrobial silk to be applied on burn victims to combat sepsis.
Team:UCLA 2014 iGEM project <https://2014.igem.org/Team:UCLA>
Team:UCLA 2015 iGEM project <https://2015.igem.org/Team:UCLA>
Dove A (2002) Uncorking the biomanufacturing bottleneck. Nature Biotechnology 20 (8): 777-779. DOI: 10.1038/nbt0802-777
Lewis R (2006) Spider Silk: Ancient Ideas for New Biomaterials. Chemical Reviews 106 (9): 3762-3774. DOI: 10.1021/cr010194g
Mayfield SP, Franklin SE, Lerner RA (2003) Expression and assembly of a fully active antibody in algae. Proceedings of the National Academy of Sciences 100 (2): 438-442. DOI: 10.1073/pnas.0237108100
Wijffels RH, Kruse O, Hellingwerf KJ. Potential of industrial biotechnology with cyanobacteria and eukaryotic microalgae. Curr Opin Biotechnol. 2013 Jun;24(3):405–13. 43.
