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| - | <p>You can download the pdf version, which is prettier and cooler, HERE! (link futuro aquí)</p>
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| - | <br><font color="#0174DF"><p><b>Overview</b></font></p>
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| - | <p align="justify">IGEM CIDEB 2014 considers biosafety as important as every other points of the iGEM competition. Because of this reason, the team decided to perform a Safety Risk Assessment focused on the project and in the lab practices needed to accomplish it.
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| - | In this assessment, a description of our host organism is made, along with the genetic modifications that were applied to it, including preventive measures to avoid its dissemination and appropriate identification and containment measures, in the case it was released into the environment. Also the overall potential risks of the project were included, taking in consideration all of the possible risks of working in our laboratory, along with preventive measures to reduce risk to a minimum.</p>
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| - | <br><font color="#0174DF"><p><b>Organism's description</b></font></p>
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| - | <p><i>Escherichia coli (E. coli)</i> is a large and diverse genus of bacteria belonging to the <i>Enterobacteriaceae.</i> Although most strains of <i>E. coli</i> are relatively harmless, some can potentially affect humans and animals. Pathogenic kinds of <i>E. coli</i> can cause diarrhea, along with urinary tract infections, respiratory illness and pneumonia, among other symptoms. <i>E. coli</i> can be commonly found in the digestive tract of humans and many animals. It plays an important role in the decomposition and absorption of certain nutrients in the intestine that the body cannot break down by itself and to also prevent the digestive track to be colonized by other harmful bacteria.</p>
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| - | <p><i>E. coli</i> are capable of both aerobic and anaerobic cellular respiration, which is a characteristic that allows them to live in both oxygen rich and oxygen poor environments, which has allowed them to thrive in a wide variety of ecosystems.</p>
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| - | <img width=250 height=200 src="https://static.igem.org/mediawiki/2014hs/f/f3/EcoliCIDEB.jpg" align=left hspace=12>
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| - | <p>As a prokaryote, E. coli bacterium has no organelles, and its genetic information is not enclosed in a nucleus. E. coli protective layer consists on a cell wall and a capsule that protects it from the outside, potentially harmful environment. E. coli goes through binary fusion on a regular basis if given the right conditions, usually at 37° Celsius, and it is able to thrive and reproduce at a very fast rate.</p>
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| - | <p>As previously mentioned, <i>E. coli</i> is one of the most diverse genera of bacteria, probably due to its adaptive abilities. Although there is a wide variety of different <i>E. coli</i> strains to choose from, not all of them have the same characteristics; some of them are pathogenic and are not safe to work with, which is the main reason why during the practices at the team’s laboratory, the <i>E. coli</i>’s strain that was used is the K12 DH5-α strain, which is one of the safest strains to work with, and one of the most used in biotechnology research. The K12 DH5-α strain is characterized by its poor abilities to colonize plant and animal tissue, and a low resistance to outside-lab environment, temperature fluctuation and different media composition causing the organism to die.</p>
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| - | <p><i>E. coli</i>’s K12 DH5-α inability to colonize intestinal tissue was experimented in 1978 in a work made by R. Curtiss “Biological containment and cloning vector transmissibility” showing that the K12 DH5-α strain is not likely to behave as a pathogen in mammal tissue. Due to these previous mentioned characteristics, it is classified as a Class 1 Containment under the European Federation of Biotechnology guidelines, and according to the United States Environmental Control Agency (EPA) <i>E. coli</i> K12 DH5-α strain opposes a very low risk for other organisms and under contained conditions of fermentation and are safe to work with.</p>
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| - | <br><font color="#0174DF"><p><b>Genetic modifications</b></font></p>
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| - | <p>In order to accomplish the iGEM CIDEB 2014 project’s objective, <i>E. coli</i> went through some genetic modifications. The E. CARU project is divided into four different modules, each one of them adding a different characteristic to the bacterium. The four modules are:</p>
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| - | <p> 1. Resistance</p>
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| - | <p> 2. Capture</p>
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| - | <p> 3. Aroma</p>
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| - | <p> 4. Union</p>
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| - | <br><p><b>1. Resistance module</b><p>
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| - | <img width=260 height=210 src="https://static.igem.org/mediawiki/2014hs/0/0c/ExperimentalCIDEB.jpg" align=right hspace=12>
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| - | <p>Unmodified <i>E. coli</i> K-12 is able to tolerate added salt of up to 10% concentration (M. Don, 2008), however, E. CARU was tested with higher amounts than those mentioned (For further information, check the Capture module in this wiki).</p>
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| - | <p>In order to work with abnormal higher saline concentrations without killing the bacteria, IrrE, a gene that provides resistance to some adverse conditions for it, was introduced to <i>E. coli</i>.</p>
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| - | <p>The gene IrrE up regulates the production of several stress responsive proteins, protein kinases, metabolic proteins, and detoxification proteins. It also down-regulates glycerol degradation. With this global regulatory effect, <i>E. coli</i> becomes more salt tolerant (UCL, 2012).</p>
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| - | <p>The module’s sequence is as follows:</p>
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| - | <center><img width=290 height=130 src="https://static.igem.org/mediawiki/2014hs/f/ff/ResistanceCIDEB.jpg" align=center hspace=12></center><br>
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| - | <p>The sequence begins with a constitutive promoter (BBa_J23119), followed by an RBS (BBa_B0034), the gene IrrE (BBa_K729001) and a terminator (BBa_B1002).</p>
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| - | <br><p><b>2. Capture module</b><p>
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| - | <p>One of the most important genetic modifications in the project is the capture of sodium ions in order to desalinize water. This was made possible by taking advantage of NhaS, a putative gene which is characterized after its expression, “by its corresponding protein ability to bind and sequestering sodium ions.” (Ivey, Krulwich, 1994).</p>
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| - | <p>The project’s circuit sequence is:<p>
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| - | <center><img width=310 height=130 src="https://static.igem.org/mediawiki/2014hs/8/82/ProjectCaptureCIDEB.jpg" align=center hspace=12></center><br>
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| - | <p>Since NhaS is putative, iGEM CIDEB 2014 decided to test the module with a red fluorescence protein (BBa_E1010), which is simpler than the original reporter idea for the module, and this allowed us to test one gene at a time in each module.</p>
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| - | <p>The sequence used for the NhaS experimentation is:</p>
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| - | <center><img width=310 height=130 src="https://static.igem.org/mediawiki/2014hs/5/54/ExperimentCaptureCIDEB.jpg" align=center hspace=12></center><br>
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| - | <p>An UV Promoter (BBa_I765001) was chosen to begin the circuit in order to control the NhaS gene’s expression in E. CARU. The promoter is followed by an RBS (BBa_B0034), the NhaS gene (BBa_K1255000), an RFP reporter (BBa_E1010) and a terminator (BBa_B1002).</p>
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| - | <p>Basically the same, just changing the RFP reporter for BSMT1 Opt (BBa_K1255001), which is the CDS that is able to produce a Wintergreen aroma. For further information look at the Aroma module in this document.</p>
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| - | <br><p><b>3. Aroma module</b><p>
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| - | <p>The use of reporters differing from the usual fluorescence proteins is one of the objectives iGEM CIDEB 2014 team is pursuing by using aromatic reporters, like banana or, in this case, wintergreen odor.</p>
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| - | <p>The aroma module is used in order to prove the effectiveness of BSMT1 Opt CDS (BBa_K1255001), for further use as an odor reporter for other teams and modules for this project. </p>
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| - | <center><img width=290 height=130 src="https://static.igem.org/mediawiki/2014hs/4/4c/AromaCIDEB.jpg" align=center hspace=12></center><br>
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| - | <p>BSMT1 (Salicylic Acid Carboxyl Methyltransferase I) is formed as part of a different circuit, composed by a constitutive promoter (BBa_J23119), a riboswitch (RNA thermometer, BBa_K115017), a CDS that, when it is induced by salicylic acid, it releases an enzymatic product (methyl salicylate), responsible of wintergreen odor, and a terminator (BBa_B1002).</p>
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| - | <img width=270 height=220 src="https://static.igem.org/mediawiki/2014hs/1/16/WgCIDEB.jpg" align=left hspace=12>
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| - | <p>This sequence will help to test its effectiveness and future usage as an odor reporter, since other teams (MIT 2006) have just analyzed it theoretically. IGEM CIDEB 2013 uses a riboswitch to induce the gene expression at high temperatures.</p>
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| - | <p>This piece (BSMT1 Opt) can replace RFP on capture module, or be added on union module; as wintergreen odor to demonstrate the presence of bacteria in silica beads or the capture of sodium ions on salty environments.</p>
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| - | <p>This module will be tested on a culture medium, and induced by salicylic acid to produce WG (WinterGreen) odor.</p>
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| - | <br><p><b>4. Union module</b><p>
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| - | <p>The main objective for iGEM CIDEB 2014 team is the construction of a biological circuit capable to capture sodium ions, and to remove them by using a silica-beads based bio-filter. In this module, the outer membrane of the bacteria is modified so it can bind silica or glass surfaces.</p>
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| - | <center><img width=290 height=130 src="https://static.igem.org/mediawiki/2014hs/7/79/UnionCIDEB.jpg" align=center hspace=12></center><br>
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| - | <img width=150 height=200 src="https://static.igem.org/mediawiki/2014hs/8/88/BiofilterCIDEB.jpg" align=right hspace=12>
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