Team:Hong Kong-CUHK/project-2.html

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<h2>Introduction</h2>
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        <h2>PAH Degradation</h2>
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     Carbon dioxide (CO2) is notorious for its major contribution to global warming, where one of the impacts brought to the ecosystem is its excessive solvation into the ocean in carbonate form, threatening marine lifes (Baldgcchi et al., 1996). This year we would like to utilize and recharge these abundantly available CO2 by converting it to methane (CH4), an important carbon source for fuels and bio-degradable plastic production. While there are naturally existing methane-generating microorganisms, the convertion process involves multi-step metabolic reactions, not to mention that the mircoorganisms can only survive in anaerobic environment. Therefore, the difficulty of manipulating this convertion process remains high.
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    A recent research showed that a mutated form of nitrogenase from Azotobacter vinelandii, a nitrogen-fixing bacteria found in soil, has carbon fixation ability (Seefeldt et al., 2013). Yang et al. demonstrated that by introducing 70Ala and 195Gln mutations on nitrogenase alpha subunit, the nitrogenase enzyme complex reduced CO2 and CO32- to CH4 instead of converting N2 to NH3 (Yang et al., 2012). This system provided an one-step reaction to convert CO2 into CH4 and other carbon compounds directly. However, since a large electron flux, and thus energy, was wasted in producing molecular hydrogen (H2) from protons during the reaction, we utilized a soluble hydrogenase complex from Aquifex aeolicus to recycle H2 to protons. To further enhance the efficiency of carbon fixation process, we physically linked both nitrogenase and hydrogenase complexes with SH3 and PDZ ligand-domain pairs to accelerate the H2 recycling.
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            <h3>Background</h3>
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            <p>Our project initiated from dealing with toxic chemicals found in second-hand smoke (or environmental tobacco smoke, ETS) and cooking fume. After carefully examining the composition of pollutes, we noticed that there was one group of highly toxic and carcinogenic compounds, polycyclic aromatic hydrocarbons (PAHs). The toxicity of PAHs is difficult to handle, and many scientists are doing intensive research on this issue.</p>
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            <p>PAHs are notorious for its harm to both environment and human health. PAHs commonly appear in people’s daily life, from second-hand smoke to cooking fume. Also, PAHs can be generated in a large amount by incomplete combustion of carbon-containing fuels. (Li et al. 2003, Zhang et al. 2013) PAHs may dissolve in air, diffuse in water, or precipitate in soil, causing large-scale, wide-spread pollution. (Samanta, Singh, and Jain 2002).Furthermore, most of the PAHs is recorded to be carcinogenic, mutagenic, or teratogenic. (Li et al. 2003, Gauggel-Lewandowski et al. 2013) For example, the first and foremost study of carcinogenicity of benzo[a]pyrene (BAP), a compound belongs to PAHs family, dates back to the observation that chimney sweepers developed scrotum cancer easier than others in 18th century. (Boffetta, Jourenkova, and Gustavsson 1997)</p>
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            <p>With the power of synthetic biology, we aimed to develop a method using E. coli to degrade PAHs into other non-toxic chemicals.</p>
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            <h3>Degradation Pathway</h3>
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            <p>To degrade PAHs, we used a combination of two different codon-optimized enzymes, namely laccase from
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                <em>Bacillus sp.</em>HR03 and catechol 1,2-dioxygenase from
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                <em>Pseudomonas putid</em>a KT2440, in
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                <em>Escherichia coli</em>. After steps such as oxidation and ring-cleavage, PAHs can be degraded into more simple and less toxic chemicals (figure: proposed degradation pathway).</p>
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            <p>&nbsp;</p>
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            <p align="center">
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                <img src="https://static.igem.org/mediawiki/2013/0/05/Pah5.png" alt="5" width="591" height="194">
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                <strong>Laccase</strong>
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                <br>Laccase involved in the first step of degradation pathway. Hadibarata
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                <em>et al</em>.[2] proposed that laccase could play a significant role in benzo[a]pyrene (BaP) degradation. It was suggested that laccase would add oxygen atoms onto the ring, changing PAHs into quinone-like intermediates, which are easier for degradation, due to their higher water solubility and weaker phenolic structures [2, 9]. Among various bacterial laccases, we chose the laccase from
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                <em>Bacillus sp</em>. HR03. The optimal conditions for this laccase are pH7 and 70 C, with acceptable activity in lower temperature such as 20 ºC [6]. Therefore, it is suitable for our experiment.</p>
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                <strong>Catechol 1,2-dioxygenase</strong>
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                <br>Followed by the action of laccase, catechol 1,2-dioxygenase from
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                <em>Pseudomonas putida</em>KT240 continues further degradation on the quinone-like compound [5]. Dioxygenase is an oxidative enzyme which induces ortho-ring cleavage of catechol, a phenolic intermediate of several metabolisms related to aromatic compound degradations [1, 5]. Moreover, a previous study indicated that 1,2-dioxygenase may play an important role in BaP degradation [2]. Therefore, this enzyme was included for the proposed metabolic pathway of PAHs in the project. The optimal conditions for catechol 1,2-dioxygenase are pH7.5-8 and 25-30 ºC [5].</p>
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                <strong>QsrR</strong>
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                <br>To regulate PAHs degradation, we used quinone sensing and response repressor (QsrR).&nbsp;QsrR is a transcriptional regulator under the thiol-stress-sensing regulator YodB family in
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                <em>Staphylococcus aureus</em>[4]. In our proposed pathway, the intermediates, quinone-like compounds, can function as the signaling molecules. Once bind with quinone molecules, QsrR would leave the target DNA, and thus allow&nbsp;the expression of downstream enzymes for subsequent degradations. With the regulation from QsrR,
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                <em>E.coli</em>can avoid producing unnecessary enzymes under PAHs-free situation, and therefore make them more viable.
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Latest revision as of 07:15, 27 November 2014

Introduction

Carbon dioxide (CO2) is notorious for its major contribution to global warming, where one of the impacts brought to the ecosystem is its excessive solvation into the ocean in carbonate form, threatening marine lifes (Baldgcchi et al., 1996). This year we would like to utilize and recharge these abundantly available CO2 by converting it to methane (CH4), an important carbon source for fuels and bio-degradable plastic production. While there are naturally existing methane-generating microorganisms, the convertion process involves multi-step metabolic reactions, not to mention that the mircoorganisms can only survive in anaerobic environment. Therefore, the difficulty of manipulating this convertion process remains high.

A recent research showed that a mutated form of nitrogenase from Azotobacter vinelandii, a nitrogen-fixing bacteria found in soil, has carbon fixation ability (Seefeldt et al., 2013). Yang et al. demonstrated that by introducing 70Ala and 195Gln mutations on nitrogenase alpha subunit, the nitrogenase enzyme complex reduced CO2 and CO32- to CH4 instead of converting N2 to NH3 (Yang et al., 2012). This system provided an one-step reaction to convert CO2 into CH4 and other carbon compounds directly. However, since a large electron flux, and thus energy, was wasted in producing molecular hydrogen (H2) from protons during the reaction, we utilized a soluble hydrogenase complex from Aquifex aeolicus to recycle H2 to protons. To further enhance the efficiency of carbon fixation process, we physically linked both nitrogenase and hydrogenase complexes with SH3 and PDZ ligand-domain pairs to accelerate the H2 recycling.

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