Team:Stuttgart/Design
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:
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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. 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 - KERATINASESThe 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 Limonene |
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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 Limonene. ... 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 comprised four steps (Fig.1):... LIMONENE 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 turpentine-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. |
