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

 
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<p>AlgAranha Team USP_UNIFESP-Brazil</p>
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<p>AlgAranha Team USP-UNIFESP BRASIL</p>
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<p style="text-align: right; padding-right: 10.5%;"><a href="https://2016.igem.org/">iGEM 2016</a></p>
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<p style="text-align: right;"><a href="https://2016.igem.org/">iGEM 2016</a></p>
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<li><a href="https://2016.igem.org/wiki/index.php?title=Team:USP_UNIFESP-Brazil" >Home</a></li>
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<li><a href="https://2016.igem.org/Team:USP_UNIFESP-Brazil/Team">Team</a></li>
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<li><a href="https://2016.igem.org/Team:USP_UNIFESP-Brazil/Project" >Project</a></li>
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<li><a href="https://2016.igem.org/Team:USP_UNIFESP-Brazil/Interlab">Interlab</a></li>
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<li><a href="https://2016.igem.org/Team:USP_UNIFESP-Brazil/Awards">Awards</a></li>
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<li><a href="https://2016.igem.org/Team:USP_UNIFESP-Brazil/Awards">Awards</a></li>
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<li><a href="https://2016.igem.org/Team:USP_UNIFESP-Brazil/Attributions">Attributions</a></li>
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<h2> AlgAranha </h2>
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            <h2>Table of contents</h2>
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                    <p class="black"><a href="#Introduction">Introduction</a></p>
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            <h2>Introduction</h2>
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                In the twelfth iGEM edition happens the third Interlab Study. This study is based on the characterization of standard biological parts and, as standard parts, it is fundamental to observe reproducibility and repeatability on their behaviour. For instance, even well characterized promoters in a given strain of E. coli may behave reasonably different in another strain. Acknowledging this challenge, the Interlab Studies is a way to gather experiments from all around the world and provide a more unified understanding about the fundamental building blocks of Synthetic Biology. Until last year, each research team had its own strains, plasmids and protocols, however, in an attempt to standardize the obtained data, specific protocols and calibration samples were provided for each iGEM team attending the Interlab 2016. With this approach, we can construct a rich knowledge base of standard biological parts, together with several study cases of different protocols and other details. The value this have to the whole community of Synthetic Biology is beyond doubt.
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                We have done not only the standard plate reader, cuvette-based and flow cytometry assays, but also tested for better measuring conditions (LB and M9 media) and for alternative methods ranging from DIY ones (digital camera and fluorimetric-based methods) to single cell analysis by fluorescence microscopy. We have also evaluated the promoter strength of all devices by Relative Promoter Units [2] using DH5α E. coli harbouring all devices and controls. Results show interesting differences: Device 2 (J23106) shows half the strength it would be expected in the original library. Thus, we have fulfilled both the InterLab study the extra credit requirements by searching for optimized measurement protocols and generating new cheaper and accessible approaches for assessing promoter strength.
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                <p class="black"><b>Test Devices and controls:</b></p>
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                <p class="black">We have received three Test Devices and one positive control derived from the Anderson’s library, a constitutive promoter library generated by single mutations, which affected the promoters’ strength in different ways. The devices are a combination of the Anderson’s promoters, RBS, a GFP reporter gene and a terminator. The negative control consist only on an inert sequence derived from the TetR operator. All devices and controls have the pSB1C3 plasmid (high-copy number) as backbone. Following the iGEM protocol, all plasmids were transformed into DH5α E. coli cells - following the iGEM transformation protocol - which were used as samples for all the different experiments. You can find more information about the devices below and on <b>figure 1</b>.</p>
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                <p class="black"><b>Positive control (PC) - I20270 in pSB1C3 </b></p>
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                <p class="black"><b>Negative control (NC) -R0040 in pSB1C3</b></p>
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                <p class="black"><b>Test Device 1 (TD1) - J23101.B0034.E0040.B0015 in pSB1C3</b></p>
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                <p class="black"><b>Test Device 2 (TD2) - J23106.B0034.E0040.B0015 in pSB1C3</b></p>
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                <p class="black"><b>Test Device 3 (TD3) - J23117.B0034.E0040.B0015 in pSB1C3</b></p>
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                        <img src="https://static.igem.org/mediawiki/2016/0/09/TT--USP_UNIFESP-Brazil--interlab_figure1.png" style="margin-bottom:20px; margin-top:0px;"/>
 
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                <p class="black"><b>Multi-scale combined experiments:</b></p>
 
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                Imagine you are a Biohacker or someone very interested in Science stuff, but you have no money… How could you avoid expensive high-end equipment and yet, still obtain some data about the promoters you love?
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                In order to do so and draw a sketch of our promoter’s strength we have followed and updated the 2015 <a href="https://2015.igem.org/Team:Brasil-USP/interlabstudy">USP_Brazil iGEM team approach</a> for an inexpensive and quick analysis by taking digital photos and analyzing them on open-source softwares for image processing (<a href="https://www.gimp.org/">GIMP</a> and <a href="https://imagej.nih.gov/ij/">ImageJ</a>)
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                All test devices and controls were grown on both solid (LB-Agar) and Liquid (LB and M9) media and photos were taken by a regular cellphone under the effect of fluorescent white or blue light lamps (for exciting GFP reporter molecules). The choice of comparing both LB and M9 liquid media was based on an extensive number of reports regarding the influence of auto fluorescence of LB on measurements. Thus, we wanted to have check if the outcome of this effect would be so strong that it could be visually detected.
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                On a direct analysis under the blue light lamp, we can observe that there is a huge difference between M9 and LB samples (Figure 2). While we can easily observe different degrees of GFP expression on M9, it is almost impossible to do so on LB due to its intrinsic fluorescence. Even though, on both media, TD1 seems to be the strongest promoter, followed by TD2, which is similar to PC and stronger than TD3 (easier to see on M9). The last test device, TD3, seems to behave very similarly to the negative control. To sum up, at a first glance, our promoter’s strength rank is:
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                <center><b>TD1 > TD2 = PC > TD3 = NC</b></center>
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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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                        Figure 2 Comparison between fluorescence of Test Devices on both M9 and LB. While M9 allows us to easily compare fluorescence intensities the same is not true for LB samples due to its auto fluorescence effect.
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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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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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            <h2><font size='5'>Microscopic analysis/single cell:</font><font size='2'> Fluorescence Microscopy and Quantitative analysis by IMAGEJ</font></h3>
 
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<img src="https://static.igem.org/mediawiki/2016/6/6d/T--USP_UNIFESP-Brazil--chlamydomonas2-1.jpg" style="margin-bottom:20px;margin-top:0px;margin-left:180px"/>
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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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<h4> Help the team! </h4>
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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><a href=" http://go.dodofunding.com/campaigns/algaranha/">International campaign</a></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.