Difference between revisions of "Team:Stuttgart/Description"
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<br><h3><span style="color:#2E9AFE">WHATS THE PROBLEM?</span style="color:#2E9AFE"></h3> | <br><h3><span style="color:#2E9AFE">WHATS THE PROBLEM?</span style="color:#2E9AFE"></h3> | ||
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2.You remember what your mother taught you, so you put a fancy mix of vinegar, baking soda and some magic into the drain… a mysterious creature arises, but nothing else happens.<br> | 2.You remember what your mother taught you, so you put a fancy mix of vinegar, baking soda and some magic into the drain… a mysterious creature arises, but nothing else happens.<br> | ||
3.In the end there is just one thing left – you put the nasty chemical mixture of the stinky cleaning agent down the drain. The corrosive cloud disappears and you can finally take your shower… still trying not to breath in the acrid fumes.</h4> | 3.In the end there is just one thing left – you put the nasty chemical mixture of the stinky cleaning agent down the drain. The corrosive cloud disappears and you can finally take your shower… still trying not to breath in the acrid fumes.</h4> | ||
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<img src="https://static.igem.org/mediawiki/2017/0/0b/Comicstuttgart.png"/ width=480px height= 420px > | <img src="https://static.igem.org/mediawiki/2017/0/0b/Comicstuttgart.png"/ width=480px height= 420px > | ||
<h4>Pipe clogging and closure occurs in every household as well as in industry. Various components contribute to the pipe closure, such as fat and hair. Commercial chemical pipe cleaners contain aggressive substances, which are harmful to the environment and health and also can lead to pipe breaks. Biological alternatives (purified enzymes) have been researched for years, without reaching the efficiency of chemical cleaners. In addition, the purification of the enzymes is very complex and cost intensive. </h4> | <h4>Pipe clogging and closure occurs in every household as well as in industry. Various components contribute to the pipe closure, such as fat and hair. Commercial chemical pipe cleaners contain aggressive substances, which are harmful to the environment and health and also can lead to pipe breaks. Biological alternatives (purified enzymes) have been researched for years, without reaching the efficiency of chemical cleaners. In addition, the purification of the enzymes is very complex and cost intensive. </h4> | ||
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<br><h3><span style="color:#2E9AFE">OUR SOLUTION</span style="color:#2E9AFE"></h3> | <br><h3><span style="color:#2E9AFE">OUR SOLUTION</span style="color:#2E9AFE"></h3> | ||
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<h1 align=middle > LIGHT UP THE PIPE - THREE PARTS FOR A BETTER FLOW </h1> | <h1 align=middle > LIGHT UP THE PIPE - THREE PARTS FOR A BETTER FLOW </h1> | ||
| − | <br>< | + | <br><h2>For the cleaning and degradation process of clogged drains by e.coli we created a genetic circuit with different enzymes (model Link). Three parts are needed for a better flow: </h2> |
Revision as of 10:56, 21 October 2017
project description
WHATS THE PROBLEM?Our project deals with an everyday problem described by the following scenario:
You want to enjoy a refreshing shower in the morning, but your hairy roommate clogged the drain again? You would like to have a relaxed bubble bath after a long day, but there are bad odors coming out of the pipe system?
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LIGHT UP THE PIPE - THREE PARTS FOR A BETTER FLOW
For the cleaning and degradation process of clogged drains by e.coli we created a genetic circuit with different enzymes (model Link). Three parts are needed for a better flow:
PART I - ESTERASES and LIPASESHair is surrounded by a layer of grease and waxes which first need to be removed to make the hair-keratin available for keratinases. For the first degradation step we choose a combination of esterases and lipases. We investigated two different esterases for their enzyme activity. One esterase from the registry (EstCS2 BBa_K1149002) and one esterase (LipB) supplied by Dr. Eggert from Evoxx were compared. Additionally we choose the lipase TliA to support the esterases at the fat degradation and to accelerate the entire degradation process. For the extracellular secretion of the enzymes we attached different signal sequences (pelB, OmpA and phoA) that were provided on the iGEM plates. EstCS2EstCS2 from the iGEM Imperial College 2013 was proved to be active. In their project the cells expressing these construct were grown and lysed by sonication and were utilized in a colourimetric assay with the substrate analog para-Nitrophenyl butyrate. In our project we didn’t purify the esterases but used the supernatant for the enzyme activity assay.LipBLipB showed an enzyme activity of 2,8 U/mL in the supernatant. First, we repeated the enzyme activity assay from the iGEM TU Darmstadt 2012 to determine the esterase with the highest enzyme activity.TliAThe lipase that is used for this project is the TliA lipase from Pseudomonas fluorescens. TliA is secreted by the ABC (ATP binding cassette) export system (prtDEF gene cluster) from Dickeya dadantii (formerly known as Erwinia chrysanthemi). The LARD secretion tag for the ABC export system is added directly to the TliA gene, which hopefully leads to a good export yield and a high extracellular activity. In order to control gene expression, the TliA gene is expressed by the pBAD promoter, which can be induced by arabinose. The prtDEF gene cluster is expressed by a constitutive promoter of mediocre strength. So, the ABC export system is always expressed at a certain level and can export the LARD-tagged TliA lipase, if expressed. |
PART II - KERATINASESFor efficient hair degradation we chose different keratinases (KerUS, KerA, KerBL and KerP). Moreover we combined this keratinases with different anderson-promotors (BBa_K206000, BBa_J23114 and BBaJ23102) and signal sequences (PelB, OmpA and PhoA) for extracellular transport of the keratinases. |
PART III - A LOVELY SCENT OF ...The microbial synthesis of natural flavor compounds has become a very attractive alternative to the chemical production (1). In recent years microorganisms such as E.coli and Yeast have been metabolically engineered to produce different flavors like limonene, geraniol or rose (1,2,3). For our project we discussed different approaches and choose two different scents: rose and fir. ... ROSE FRAGRANCEAs first special fragrance we want to install a lovely scent of rose in our microbial system. Hair are commonly made of Keratin (90%) and small amounts of amino acids, such as L-phenylalanine. This amino acid can be used as substrate for the production of 2-Phenylethylacetate (2-PEAc), which has a rose-like odor (1). Therefor this odor can act as an indicator for keratin degradation. In recent studies from Guo et al the 2-PEAc biosynthetic pathway was successfully designed and expressed in E.coli (1). This pathway was used for our project and comprised four steps (Fig.1):
... FIR FRAGRANCELimonene is a well-known cyclic monoterpene which can occur in two optical forms (2). (D)-Limonene is one of the most important and widespread terpenes in the flavor and fragrance industry, for example in citrus-flavored products such as soft drinks and candy (2). The (L)-Limonene form has a more harsh fir-like odor with a lemon-note (2). For our project we choose an enzyme-cascade, beginning with acetyl-coA and leading to the product (L)-limonene. This biosynthetic pathway was designed and inserted in E.coli (Fig.2)
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REFERENCES
