Difference between revisions of "Team:USP UNIFESP-Brazil/Project"

The protein structure of spider silk is composed of two constant terminal domains and a variable middle structural domain. Our design uses the substitution of this middle domain by a protein of interest, as used by Team:UCLA in 2014. In our case, we are using enzybiotics.

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 (Tokareva et al. 2014). In addition, terminal domains (N-terminal NT and C-terminal CT) are highly conserved both among species and different types of silk (Garb et al. 2010), which suggests they play important roles in the formation of silk and not in the generation of its mechanical properties per se. So the integration of the enzybiotic sequence to silk by flanking it with NT and CT should make the proteins to be polymerized along with the structural silk proteins (MaSp) when both are expressed.

The design for the plasmids can be separated in two “blocks”: an algae expression vector and, as the GOI (gene of interest) region in the vector, the sequence for the protein we want to put into the silk, along with NT and CT sequences. This protein coding sequence is flanked with Xho l and Bam HI. We optimized the codons for the expression in C. reinhardtii nucleus (Fuhrmann et al. 1999) and also inserted rubisco introns in the promoter hsp70A/rbcs2 sequence, in the Sh-ble sequence and in the terminal region RbcS2 3’ UTR, aiming to increase the expression of the protein of interest (Eichler-Stahlberg et al. 2009, Lumbreras et al. 1998). Fig. 3 shows the generic cassette for expression.

In our project, we propose to explore the modular characteristic of spider silk proteins, by using it as an immobilization support to other proteins. We were inspired by UCLA’s iGEM Team’s project in 2014 and 2015, where they presented the idea of using silk fibers to integrate other functional proteins to the silk’s structure. We tried to expand on this concept by expressing proteins with antimicrobial activity, enzybiotics (Fig.1). By combining these proteins and their properties, we tried to tackle a major problem with wound dressings for burn victims.

We tried to express the recombinant proteins, spider silk proteins and enzybiotics in the microalgae Chlamydomonas reinhardtii strains by nuclear transformation. Each recombinant strain would express a different protein, which would contain the N- and C-terminal polymerization domains from native spider silk proteins. These domains are essential to the polymerization step and, subsequently, for production of a material very similar to silk. Having been able to build our design, the antimicrobial activity and mechanical properties of the product would be evaluated, as well as the system productivity, shedding some light on spider silk-based immobilization support effectiveness, even for other biotechnological applications, such as the one idealized here. However, there are other possible applications with economic and academic interest.

Microalgae present various desirable characteristics in an expression system: fast growth, fast making of stable transgenic lineages, scalability and low production cost, for example ​(Wijffels 2013, Rosenberg 2008)​. Unlike bacterial expression systems, microalgae are capable of producing and secreting complex proteins with post-transcriptional modifications. Mammalian cells also wouldn’t be an optimal expression system when considering production costs. Molecules such as monoclonal antibodies (mAbs) are mainly produced in these cells and their average production cost in this system is estimated to be $ 150.00 per gram of raw materials (Dove 2002), but the estimated value for algae reaches US $ 0.002 per liter, making them potential competitors (Mayfield et al. 2003). Another problem with spider silk expression is the G-C rich content of its sequences, often clogging the heterologous expression of this kind of protein in non-GC-rich systems (Yang et al. 2016). But Chlamydomonas reinhardtii presents a GC-rich genome, which may play an important role in spider silk protein expression.

Immobilization techniques are applied to a wide range of treatments and processes, from medical applications to biotransformations in industrial plants. This stabilization is normally achieved by protein binding to a scaffold (Liese and Hilterhaus 2013). Recent studies explored spider silks as a possible immobilization support (Blüm et al. 2013, Monier 2013). 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 (Lewis 2006). Furthermore, medical applications are possible due to its biocompatibility and biodegradability, as coating for implants and transplanted organs, drug delivery and scaffolding for cell lines (Lewis 2006, Hardy et al. 2008, Kluge et al. 2008).

Soon…

Team:UCLA 2014 iGEM project <https://2014.igem.org/Team:UCLA>

Team:UCLA 2015 iGEM project <https://2015.igem.org/Team:UCLA>

Blüm C, Nichtl A, Scheibel T (2013) Spider Silk Capsules as Protective Reaction Containers for Enzymes. Advanced Functional Materials 24 (6): 763-768. DOI: 10.1002/adfm.201302100

Dove A (2002) Uncorking the biomanufacturing bottleneck. Nature Biotechnology 20 (8): 777-779. DOI: 10.1038/nbt0802-777

Hardy J, Römer L, Scheibel T (2008) Polymeric materials based on silk proteins. Polymer 49 (20): 4309-4327. DOI: 10.1016/j.polymer.2008.08.006

Kluge J, Rabotyagova O, Leisk G, Kaplan D (2008) Spider silks and their applications. Trends in Biotechnology 26 (5): 244-251. DOI: 10.1016/j.tibtech.2008.02.006

Lewis R (2006) Spider Silk: Ancient Ideas for New Biomaterials. Chemical Reviews 106 (9): 3762-3774. DOI: 10.1021/cr010194g

Liese A, Hilterhaus L (2013) Evaluation of immobilized enzymes for industrial applications. Chemical Society Reviews 42 (15): 6236. DOI: 10.1039/c3cs35511j

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

Monier M (2013) Immobilization of β-galactosidase from Escherichia coli onto modified natural silk fibers. Journal of Applied Polymer Science 130 (4): 2923-2931. DOI: 10.1002/app.39475

Rosenberg JN, Oyler GA, Loy W, Betenbaugh MJ. A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution. Curr Opin Biotechnol. 2008;19(5):430–6.

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.

Yang X-Y, Li C-R, Lou R-H, Wang Y-M, Zhang W-X, Chen H-Z, Huang Q-S, Han Y-X, Jiang J-D, You X-F (2007) In vitro activity of recombinant lysostaphin against Staphylococcus aureus isolates from hospitals in Beijing, China. Journal of Medical Microbiology 56 (1): 71-76. DOI: 10.1099/jmm.0.46788-0