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| − | <h2> Project Description </h2>
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| − | <h3> SDSeeker: Bioremediation of Lago de Guadalupe </h3>
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| − | Lago de Guadalupe is the most extensive water body in the State of Mexico. It is surrounded by industrial, agricultural and residential areas, the last one being responsible for 25% of the total contaminants, among these is SDS (sodium dodecyl sulfate), which is the principal component of detergents used in households. The residues of detergents change the water's pH, turning it into a toxic and dangerous environment for more than 150 species that depend on this lake. Although this lake has been studied for its treatment and to obtain better levels of water quality, nothing has been achieved. Our project will consist in developing a bioremediation system using synthetic biology, to lower SDS levels in the water.
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| − | The early stages of this project include capturing SDS molecules with a protein called ferritin and the catalysis of the first step in the SDS degradation pathway, mediated by an alkyl sulfatase.
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| − | <h2>Lago de Guadalupe contamination</h2>
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| − | Lago de Guadalupe is an artificial lake of 400 hectares located in State of Mexico and is also the largest water body in this area. It is located in the northwest part of the city and it is the habitat of more than 150 species of animals. <br>
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| − | The lake is located in three different zones and each one contributes to the contamination of the lake due to many factors, including home residues and industrial wastes. Some studies confirm that only 26% of the residues are treated and the rest are disposed in many lakes and water bodies causing not only their contamination, but also eutrophication (enrichment of an ecosystem because of the accumulation of nitrogen and phosphorus). Even though mexican organizations and government associations try to keep the water quality in certain levels and try to protect this lake, different studies show high levels of water contamination due to urban residues release (up to 15 million m3 per year), specially in Bosques del Lago, which is the residential zone next to Lago de Guadalupe. Nowadays it is estimated that more than 25% of the total volume of the lake are residues that contribute to its contamination. <br>
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| − | The contamination issues that can be found in this lake are the following:<br>
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| − | 1. High eutrophication.
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| − | 2. Trophic values of 87.4 +/- 7.4
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| − | 3. Low water quality. Value corresponding to “highly contaminated”: 26.0 +/- 12.3
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| − | 4. Absence of dissolved oxygen below 3 meters
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| − | 5. Pollutants’ concentrations above the standards of mexican official norms
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| − | 6. High levels of fecal coliforms presence in water <br><br>
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| − | All of the previously mentioned issues don’t let the lake maintain a stable fish population, altering the whole cycle that involves the organisms that live in this lake and directly or indirectly affect other animals in the surroundings. This situation is terribly altered by the growth in the human populations that surrounds Lago de Guadalupe. <br>
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| − | It is important that even with the help of synthetic biology and technology, the population that surrounds this lake understands what is happening and how they can be directly affected by the high levels of contamination. <br>
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| − | Lago de Guadalupe have a highest concentration of pollutants and the reason that we wanted to make our project on this lake is because we all live in nearby areas and it affects us directly, even so, water pollution is a problem that concerns every one of us!<br> </p>
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| − | <h2>SDS</h2>
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| − | <p>One of the pollutants that can be found in the residential area next to this lake is detergents.
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| − | The detergents residues are often disposed in this lake, provoking changes in the water’s pH and therefore, affecting life in this water body.<br>
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| − | Approximately 40% of the detergents content used in this area, are anionic surfactants. The most widely and commonly household detergent used is known as Sodium Dodecyl Sulfate (SDS). This compound is toxic to aquatic animals, including fish and microorganisms like yeast and some bacteria. Toxicity extends to mammals and animals that are part of the circle of life around this lake.</p>
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| − | <h2> SDS biorremediation</h2>
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| − | <p>
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| − | To reduce the adverse effects produced by SDS contamination in water bodies, investigations and protocols for its bioremediation have been established in the past years.<br>
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| − | However, most of them stayed in the strain characterization phase of detergent-degrading bacteria. When identifying certain strain that is capable of doing this, researchers found themselves with the risk of discharging high amounts of these bacteria and also altering the environment within the water bodies.<br>
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| − | This is why our team decided to develop this project using a different approach, where we can use the specific enzymes to degrade this compound.<br>
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| − | The early stages of this project include the detection of detergent presence in Lago de Guadalupe and the usage of an enzyme that is able to catalyze the first step in SDS degradation (alkyl sulfatase from <i>Pseudomonas sp.</i>) and another enzyme (human ferritin) that is capable of binding SDS molecules.
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| − | <h1>SDSeeker</h1>
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| − | <h3>Biosensor: First Step</h3>
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| − | Since its discovery, the green fluorescent protein has been used as a reporter because of its several characteristics, but mainly because is really easy to detect its expression. GFP presence can easily be detected through the use of UV at certain wavelength and is one of the most used proteins in biosensors.
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| − | <br>In the last years, the pollution levels have increased, specially in water bodies, which are reported to be contaminated by particles such as heavy metals, industrial wastes, SDS and others. SDS is an anionic surfactant and is part of most of the detergents used in Mexico. This molecule is highly toxic to aquatic environment altering the metabolism and elevating death range. The contamination can also reach mammals and humans that are in contact with contaminated water bodies causing multiple cell damage and due to this, the development of a new way to detect SDS molecules has emerged. Because there isn’t a large number of biosensors reported, many other techniques have been used to detect the presence of the molecule in water samples such as HPLC and UV. Because UV light is easy to get and easier to handle, we developed a lab scale biosensor based in detection of fluorescence of the green fluorescent protein.
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| − | <br>
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| − | The team developed a model prototype which will contain the necessary machinery to capture and degrade SDS molecules contained in water, but first we need to determine whether the water has alarmant levels of SDS or not. That is why we also developed a biosensor with the Device 3 of Interlab study which was the one that worked better and had more fluorescence emission. The biosensor will not be included in the biofilter and will consist in an apart unit of detection. <br><br>
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| − | The biosensor will work with fluorescence detection. This mechanism will be held in an agarose matrix where, immobilized E. coli will be constantly expressing the green fluorescent protein until the SDS reaches the concentration at which fluorescence will decrease. <br>
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| − | The biosensor was made first in lab scale, filling petri dishes with agarose, after the agarose solidified, SDS was added in 5 different concentrations (10%, 1%, 20 ppm, 10 ppm and 5 ppm) to observe the change of fluorescence. Then a fresh culture of modified <i>E. coli</i> (containing GFP) was plated in the dish and then incubated 37°C for 10 hours. After that time, the petri dishes were observed in a 240 nm UV lamp and obtained the following results:
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| − | <h3>Capture of SDS molecules: Second Step</h3>
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| − | <h3>Degradation: Third Step</h3>
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| − | <p>The SDS degradation pathway is very well known and starts with the enzyme alkyl sulfatase, which cleaves the sulfate group of SDS and produces the compound 1-dodecanol, a C12 alcohol that is then catalyzed into other compounds by other enzymes.
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| − | For now, we will focus in the usage of the enzyme alkyl sulfatase alone, by introducing the necessary genetic information into <i>E.coli</i> and over-producing this enzyme to characterize the first step in SDS degradation.<br>
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| − | As it has been told before, the Alkyl Sulfatase is the enzyme on charge of the first step of the degradation process of SDS. <i>Pseudomonas sp.</i> contains the gene SDSa and SDSb that is capable of forming a complex and make the conversion of SDS into alcohol.
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| − | In most cases this enzymes will catalyze the hydrolyzation of the ester bonds, this leads to the formation of inorganic sulfates that consequently are degraded or incorporated to the intracellular lipids.<br>
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| − | The genes responsible for the Alkyl Sulfatase are the SDSa and the SDSb. SDSa only metabolizes ADS mostly and SDSb regulates the metabolism of the cell since it's a LysR. The SDSa and SDSb are both controlled by the SDSb, without the SDSb the SDSa will not work. Also without the SDSb the cell will die in any medium that has SDS or something similar.<br>
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| − | SDSb has the characteristic of giving the ability to the cell to use SDSb as a carbon source. It will be able to grow at a 4% of SDS concentration and it will decrease at a 85% approximately.<br>
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| − | The alkyl sulfatase will cleave the sulfate groups into 1-Dodecanol and after that it will be oxidized by an alcohol dehydrogenase turning it in 1-Dodecanoic acid. Then it will enter into the an oxidation pathway, for its future usage as a carbon source.
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| − | <br>
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| − | We designed the sequence for synthesis of the sequence of SDSA1, which codifies for the enzyme and even though we didn't achieve to characterize the standardized enzyme of alkyl sulfatase,the enzyme itself is well characterized for the step it catalyzes, we only have to prove it with the IPTG induction in pSB1C3.
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| − | </p>
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| − | <h4>References</h4>
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| − | <p>
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| − | Sepulveda, J., Hoyos, J., Gutierrez, F.J., et. al. (2013) The impact of anthropogenic pollution on limnological characteristics of a subtropical highland reservoir “Lago de Guadalupe”, Mexico.
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| − | <br><br>
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| − | Semarnat. (n.d.) Clasificación de tensoactivos. Retrieved from: http://www.semarnat.gob.mx/archivosanteriores/temas/gestionambiental/Materiales%20y%20Actividades%20Riesgosas/sitioscontaminados/GTZ/E-Clasificacion%20de%20Tensoactivos.pdf
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| − | <br><br>
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| − | UniProt. (2015) Alkyl Sulfatase. Retrieved from: http://www.uniprot.org/uniprot/Q52556
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| − | <br><br>KEGG. (2015) Enzyme 3.1.6.19 Retrieved from: http://www.genome.jp/dbget-bin/www_bget?ec:3.1.6.19
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| − | <br><br>BRENDA. (2015) Information on EC 3.1.6.19 - R-specific secondary-alkylsulfatase. Retrieved from: http://www.brenda-enzymes.org/enzyme.php?ecno=3.1.6.19
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| − | <br><br>
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| − | Hagelueken, G., Thorsten, A., Wiehlmann, L., et. al. (2006) The crystal structure of SdsA1, an alkylsulfatase from Pseudomonas aeruginosa, defines a third class of sulfatases. Retrieved from: http://www.researchgate.net/publication/7096154_The_crystal_structure_of_SdsA1_an_alkylsulfatase_from_Pseudomonas_aeruginosa_defines_a_third_class_of_sulfatases
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| − | <br><br>
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| − | European Nucleotide Archive. (2015) Pseudomonas sp. Alkylsulfatase. Retrieved from: http://www.ebi.ac.uk/ena/data/view/AAA25989
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| − | <br><br>GenBank. (2015) Pseudomonas sp. (strain ATCC 19151) sdsA gene and 11 kd protein, complete cds's; sdsB gene, partial cds. Retrieved from: http://www.ncbi.nlm.nih.gov/nucleotide/151550?report=genbank&log$=nucltop&blast_rank=1&RID=PU6280GR014
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| − | <br><br>Maddocks, S.E., and Oyston, P.C. (2008) Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. Retrieved from: http://www.ncbi.nlm.nih.gov/pubmed/19047729
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| − | <br><br>Jovcic, B., Venturi, V., Davison, J., et. al. (2010) Regulation of the sdsA alkyl sulfatase of Pseudomonas sp. ATCC19151 and its involvement in degradation of anionic surfactants. Retrieved from: http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2010.04738.x/full
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| − | <br><br>MPBiomedicals. (2014) Methyl green. Retrieved from: http://www.mpbio.com/product.php?pid=04806404
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| − | Baker, J. and Williams, E. (1973) The use if methyl green as a histochemical reagent. Retrieved from: http://jcs.biologists.org/content/s3-106/73/3.full.pdf
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| − | <br><br>(n.a.) (2005) pH measurements - indicators. Retrieved from: http://www.ph-meter.info/pH-measurements-indicators
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| − | State University of New York at Oneonta. (2011) Spectrophotometry. Absorption measurements and their application to quantitative analysis. Retrieved from: http://employees.oneonta.edu/kotzjc/LAB/Spec_intro.pdf
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| − | <br><br>
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| − | Chaturvedi, V., & Kumar, A. (2010). Bacterial utilization of sodium dodecyl sulfate. International Journal of Applied Biology and Pharmaceutical Technology, 1(3). Retrieved June 5, 2015, from http://ijabpt.org/applied-biology/bacterial-utilization-of-sodium-dodecyl-sulfate.pdf.<br><br>
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| − | Sodium lauryl sulfate. (2011). Retrieved June 6, 2015, from http://www.drugbank.ca/drugs/DB00815.<br><br>
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| − | Lauril sulfato sódico. (2010). Retrieved June 6, 2015, from http://www.acofarma.com/admin/uploads/descarga/1638-2678f6c4b3f8011c1dc948f03c32eda620e34983/main/files/Lauril_sulfato_sodico.pdf.<br><br>
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| − | 1-Dodecanol. (2008). Retrieved June 6, 2015, from http://www.lookchem.com/1-Dodecanol/.
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