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

 
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<p style="text-align: right;"><a href="https://2016.igem.org/">iGEM 2016</a></p>
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Unfortunately, we were not able to achieve the end goal of silk production in <i> Chlamydomonas reinhardtii</i>, but we managed to do some nice things (and get a SILVER MEDAL!)
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Here you can find stuff about:
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Our <a href="https://2016.igem.org/Team:USP_UNIFESP-Brazil/Hardware">DIY Centrifuge</a>
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Our efforts to assemble the spider silk genes at the USER multimerization part of our <a href="https://2016.igem.org/Team:USP_UNIFESP-Brazil/Notebook/">lab notebook</a>
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Transforming <i>Chlamydomonas reinhardtii</i> to produce heterologous proteins, with <a href="https://2016.igem.org/Team:USP_UNIFESP-Brazil/Proof">results</a> and <a href="https://2016.igem.org/Team:USP_UNIFESP-Brazil/Notebook">protocols</a>
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If you're interested in contacting us, don't hesitate! Whether it's answering questions, just chatting (about SynBio or not, your pick!), making plans for the future or inviting us to go grab a coffee, we would be happy to reply. You can do so at our <a href ="https://www.facebook.com/iGEMUSPUNIFESP2016/">Facebook page</a>or by emailing us at <u><b>igemsp2016@gmail.com</u></b>. Checkout our <a href ="https://www.youtube.com/watch?v=i5yGrCJ7awo">campaign video</a>!
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<p class="black">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.</p>
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We are a multidisciplinary team from São Paulo, Brazil, with students from different majors, as architecture, biology, biomedical sciences, social sciences, and also from the universities USP, UNESP and UNIFESP. The team was originated from the synthetic biology club <a href ="https://s3.synbiobrasil.org/"> (SynBio Brasil), </a> which is an independent group that works promoting synbio and open science awareness and education. Since 2012, different club members have organized themselves to take part in iGEM competition.
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This year, our project was based on the heterologous expression of spider silk protein in the microalgae <i> Chlamydomonas reinhardtii</i>. We named it AlgAranha, a combination of the portuguese words for algae and spider. Besides the goal of producing enzybiotics and monomers of spider silk, we aim to achieve an improvement of  <i> Chlamydomonas </i> as a synbio chassis.<!--The project started when we looked at the problem of growing antibiotic resistance and started to think in ways to tackle it. We specially focused on injury related infections, for example in the case of burn victms. We devised the creation of an antibiotic patch, combining the spider silk physical properties with antibiotic enzymes (enzybiotics). We intend to express both the spider silk and chimeric enzybiotic proteins with spider silk motifs in <i> Chlamydomonas </i> and polymerize them together to form the product of interest. We hope to accomplish, besides the final goal of patch development, improvement of  <i> Chlamydomonas </i> as a synbio chassis and analysis of its capability of producing enzybiotics and monomers of spider silk.--> Moreover, the team is involved with open hardware developement and promotion and synthetic biology popularization, helping to promote the synthetic biology culture in Brazil, raising awareness and engaging the public.     </p>
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<p class="black">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.</p>
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<p class="black">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.</p>
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Scanning electron microscope image, showing <i>Chlamydomonas reinhardtii</i>, a unicellular flagellate used as a model system in molecular genetics work and flagellar motility studies. Author: Dartmouth Electron Microscope Facility, Dartmouth College
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<p class="black">Team:UCLA 2014 iGEM project &gt;https://2014.igem.org/Team:UCLA&gt;</p>
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<p class="black">Eichler-Stahlberg A, Weisheit W, Ruecker O, Heitzer M (2009) Strategies to facilitate transgene expression in Chlamydomonas reinhardtii. Planta 229 (4): 873-883. DOI: 10.1 007/s00425-008-0879-x</p>
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<p class="black">Fuhrmann M, Oertel W, Hegemann P (1999) A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii+. The Plant Journal 19 (3): 353-361. DOI: 10.1046/j.1365-313x.1999.00526.x</p>
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<p class="black">Garb JE, Ayoub NA, Hayashi CY (2010) Untangling spider silk evolution with spidroin terminal domains. BMC Evolutionary Biology 10 (1): 243. DOI: 10.1186/1471-2148-10-2 43</p>
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<p class="black">Tokareva O, Jacobsen M, Buehler M, Wong J, Kaplan D (2014) Structure–function– property–design interplay in biopolymers: Spider silk. Acta Biomaterialia 10 (4): 1612-1626. DOI: 10.1016/j.actbio.2013.08.020</p>
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<p><a href="http://www.vakinha.com.br/vaquinha/brasileiros-pesquisam-supercurativo-para-vitimas-de-queimaduras">Brazilian campaign</a></p>
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<p class="black">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.</p>
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<p class="fig-label">Figure 1: Schematic representation of spider silk proteins and chimeric protein. A: MaSp1 - Major ampullate spidroin 1, MaSp2 - Major ampullate spidroin 2 B: Chimeric protein of a enzybiotic with N and C terminals domains of spider silk proteins.</p>
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<p class="black">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.</p>
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<p class="fig-label">Figure 2: 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 Chlamydomonas reinhardtii and a chimeric protein envisioned in this project; and the final product, a biopatch produced from recombinant silk proteins and chimeric proteins.</p>
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<p class="black">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.</p>
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<p class="black">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).</p>
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<p class="black">Team:UCLA 2014 iGEM project &lt;https://2014.igem.org/Team:UCLA&gt;</p>
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<p class="black">Team:UCLA 2015 iGEM project &lt;https://2015.igem.org/Team:UCLA&gt;</p>
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<p class="black">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</p>
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<p class="black">Dove A (2002) Uncorking the biomanufacturing bottleneck. Nature Biotechnology 20 (8): 777-779. DOI: 10.1038/nbt0802-777</p>
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<p class="black">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</p>
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<p class="black">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</p>
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<p class="black">Lewis R (2006) Spider Silk: Ancient Ideas for New Biomaterials. Chemical Reviews 106 (9): 3762-3774. DOI: 10.1021/cr010194g</p>
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<p class="black">Liese A, Hilterhaus L (2013) Evaluation of immobilized enzymes for industrial applications. Chemical Society Reviews 42 (15): 6236. DOI: 10.1039/c3cs35511j</p>
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<p class="black">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</p>
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<p class="black">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</p>
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<p class="black">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.</p>
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<p class="black">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.</p>
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<p class="black">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</p>
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Latest revision as of 23:38, 2 December 2016

AlgAranha Team USP_UNIFESP-Brazil

iGEM 2016



Unfortunately, we were not able to achieve the end goal of silk production in Chlamydomonas reinhardtii, but we managed to do some nice things (and get a SILVER MEDAL!) Here you can find stuff about:

Our DIY Centrifuge

Our efforts to assemble the spider silk genes at the USER multimerization part of our lab notebook

Transforming Chlamydomonas reinhardtii to produce heterologous proteins, with results and protocols

If you're interested in contacting us, don't hesitate! Whether it's answering questions, just chatting (about SynBio or not, your pick!), making plans for the future or inviting us to go grab a coffee, we would be happy to reply. You can do so at our Facebook pageor by emailing us at igemsp2016@gmail.com. Checkout our campaign video!

We are a multidisciplinary team from São Paulo, Brazil, with students from different majors, as architecture, biology, biomedical sciences, social sciences, and also from the universities USP, UNESP and UNIFESP. The team was originated from the synthetic biology club (SynBio Brasil), which is an independent group that works promoting synbio and open science awareness and education. Since 2012, different club members have organized themselves to take part in iGEM competition.

This year, our project was based on the heterologous expression of spider silk protein in the microalgae Chlamydomonas reinhardtii. We named it AlgAranha, a combination of the portuguese words for algae and spider. Besides the goal of producing enzybiotics and monomers of spider silk, we aim to achieve an improvement of Chlamydomonas as a synbio chassis. Moreover, the team is involved with open hardware developement and promotion and synthetic biology popularization, helping to promote the synthetic biology culture in Brazil, raising awareness and engaging the public.