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| - | <a href="https://2013.igem.org/Team:Tianjin/Project">Background</a>
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| - | <a href="https://2013.igem.org/Team:Tianjin/Project#anchor-p-01">iGEM Handbook</a>
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| - | <a href="https://2013.igem.org/Team:Tianjin/Project#anchor-p-01">Internet Platform</a>
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| - | <a href="https://2013.igem.org/Team:Tianjin/Project#anchor-p-03">60-Second Biology</a>
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| - | <a href="https://2013.igem.org/Team:Tianjin/Project/Design">Design</a>
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| - | <ul>
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| - | <a href="https://2013.igem.org/Team:Tianjin/Project/Design#anchor-p-07">iGEM Handbook</a>
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| - | <a href="https://2013.igem.org/Team:Tianjin/Project/Design#anchor-p-08">Internet Platform</a>
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| - | <a href="https://2013.igem.org/Team:Tianjin/Project/Design#anchor-p-09">60-Second Biology</a>
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| - | <a href="https://2013.igem.org/Team:Tianjin/Human_Practice/Collaboration ">Collaboration</a>
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| - | <a href="https://2013.igem.org/Team:Tianjin/Human_Practice/Collaboration#anchor-hp-10">The 1st Model iGEM</a>
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| - | <a href="https://2013.igem.org/Team:Tianjin/Human_Practice/Collaboration#anchor-hp-11">Tsinghua University</a>
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| - | <a href="https://2013.igem.org/Team:Tianjin/Human_Practice/Collaboration#anchor-hp-12">Ocean University of China</a>
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| - | </li>
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| - | <a href="https://2013.igem.org/Team:Tianjin/Human_Practice/Collaboration#anchor-hp-13"> BIT-China</a>
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| - | </ul>
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| - | <a href="https://2013.igem.org/Team:Tianjin/Human_Practice/SocietyOutreach ">Society Outreach</a>
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| - | <a href="https://2013.igem.org/Team:Tianjin/Human_Practice/SocietyOutreach#anchor-hp-14">PetroChina Dagang Petrochemical Company</a>
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| - | </li>
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| - | <a href="https://2013.igem.org/Team:Tianjin/Human_Practice/SocietyOutreach#anchor-hp-15">Communicate with Research Institute</a>
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| - | </li>
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| - | </ul>
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| - | <a href="https://2013.igem.org/Main_Page" title="Main Page"><img src="https://static.igem.org/mediawiki/2013/7/75/IGEM-logo-blue.png" width="200px" height="200px" border="none" style="margin-top:80px;"/></a>
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| - | <center><span style="font-family:Arial;font-size:46px;color:#000;"> Background</span></center>
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| - | <a name="anchor-p-01" id=" anchor-p-01"></a>
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| | + | <a href="https://2013.igem.org/Team:Tianjin/Project/Introduction"><img src="https://static.igem.org/mediawiki/2013/d/d3/TJU-project-banner01.jpg" width="150px" height="150px" align="right" alt="Introduction" title="Introduction"/></a> |
| | + | <a href="https://2013.igem.org/Team:Tianjin/Project/Design&Construction"><img src="https://static.igem.org/mediawiki/2013/a/a2/TJU-project-banner02.jpg" width="150px" height="150px" align="left" alt="Design & Construction" title="Design & Construction"/></a> |
| | + | <a href="https://2013.igem.org/Team:Tianjin/Project/Characterization"><img src="https://static.igem.org/mediawiki/2013/9/9c/TJU-project-banner03.jpg" width="150px" height="150px" align="right" alt="Characterization" title="Characterization"/></a> |
| | + | <a href="https://2013.igem.org/Team:Tianjin/Project/Alk-Selector&DirectEvolution"><img src="https://static.igem.org/mediawiki/2013/9/98/TJU-project-banner04.jpg" width="150px" height="150px" align="left" alt="Alk-Selector & Direct Evolution" title="Alk-Selector & Direct Evolution"/></a> |
| | + | <a href="https://2013.igem.org/Team:Tianjin/Project/Futurework"><img src="https://static.igem.org/mediawiki/2013/a/a2/TJU-project-banner05.jpg" width="150px" height="150px" align="right" alt="Futurework" title="Futurework"/></a> |
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| | | | |
| - | = Call for Biofuels, especially Alkanes=
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| - | <p> Biofuels which have a closed CO2 cycle[1] and don’t require expensive, complex chemical processing, are recognized as promising replacements for diesel fuels in the fields of energy and environment. Among them, fatty-acid-derived alkanes have many advantages over other biofuel compounds, such as high caloric value and low carbon emission[2], which means that they could be an ideal replacement for diesel fuels.</p>
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| - | = Pathway of Alkane Biosynthesis=
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| - | <th> Enzymes </th>
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| - | <th>Strains </th>
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| - | <td> Acyl-ACP Reductase </td>
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| - | <td> Synechococcus elongatus PCC7942</td>
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| - | <td> Aldehyde Decarbonylase </td>
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| - | <td> Nostoc punctiforme PCC73102</td>
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| - | <p> The pathway of alkane sythesis in microbes has been studied, and heterologous expression in E.coli of the two genes encoding the two key enzymes named NPDC and AAR has been reported, which successfully turns Fatty Acyl-ACP into alkane molecules of certain chain lengths, making it possible for microbes to produce alkanes. In the strain with the highest alkane productivity, alkane titers were over 300 mg/liter using a modified mineral medium, and more than 80% of the hydrocarbons were found outside the cells[3].</p>
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| - | = Limitations of Directed Evolution=
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| - | <p> Directed evolution through random mutagenesis, followed by high-throughput selection has made it possible to improve alkane yield or pathway efficiency. However, it can be a daunting task, because there remain many constraints on throughput imposed during the standard directed evolution workflow (library construction, transformation, and screening). We consider screening and selection as rate-limiting steps in directed evolution efforts for alkane overproduction. Therefore, in the production of large chain alkanes, it is crucial for there to be a sensitive and high-throughput screening device for comparing production rates in engineered strains of E. coli.</p>
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| - | = Difficulties of Selection =
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| - | <p> Conspicuous products can be accurately measured using standard high-throughput colorimetric and fluorometric assays. Alkanes, however, like many small molecules, have no smell or fluorescence, or essential for growth, and they cannot be readily transformed into compounds possessing these properties[4]. Furthermore, commonly used screening tools like chromatography-mass spectrometry methods are inherently low throughput, with screenable library sizes generally limited to less than 103 variants. Therefore, the improved strains are beyond the reach of a general screening tool and cannot be readily obtained[5].</p>
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| - | = Use of Transcription Factors=
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| - | <p> One strategy deserving special attention is the use of transcription factors which have long been used to construct whole-cell biosensors for the detection of environmental pollutants, but remain largely untranslated toward library screening and directed evolution purposes. Transcription factors regulate a promoter’s transcriptional output in response to a small-molecule ligand so it’s possible to report on in vivo small-molecule production.</p>
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| - | <p> Screening methods utilizing transcription factors possess many ideal characteristics.The transcription factor-promoter pair can be user-selected to encode for fluorescent or growth-coupled responses, and there’s no need for downstream synthetic chemistry or in vitro manipulation [4]. Therefore,with the design of biosensors, we can transform intracellular alkane molecules without a conspicuous phenotype into detectable signal outputs.</p>
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| - | = Application of High-throughput Selection=
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| - | <p> In conclusion, utilizing high-throughput screening to select out desirable mutations is an important step in directed evolution and pathway improvement, which largely increases the library and saves labour and time. In our project, we are designing a selection module for alkanes so that we can use irrational modification to optimize the pathway of alkane synthesis.</p>
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| - | =Reference=
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| - | ----
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| - | <p>[1] Yan Kung et al. (2012) “From Fields to Fuels: Recent Advances in the Microbial Production of Biofuels.” ACS Synth. Biol. 1, 498−513</p>
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| - | <p>[2] Mathew A Rude, Andreas Schirmer et al.(2009) “New microbial fuels: a biotech perspective.” Current Opinion in Microbiology 12:274–281</p>
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| - | <p>[3] Andreas Schirmer, Mathew A. Rude et al.(2010) “Microbial Biosynthesis of Alkanes.” Science Vol. 329 no. 5991 pp. 559-562 </p>
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| - | <p>[4] Jeffrey A. Dietrich, Adrienne E. McKee, Jay D. Keasling et al.(2010) “High-Throughput Metabolic Engineering: Advances in Small-Molecule Screening and Selection.” Annu. Rev. Biochem. 79:563–90</p>
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| - | <p>[5] Jina Yang1, Sang Woo Seo1, Sungho Jang et al.(2013) “Synthetic RNA devices to expedite the evolution of metabolite-producing microbes.” Nature Communications DOI: 10.1038/ncomms2404</p>
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| - | <div class="note2"> | + | <h1><b>Overview </b></h1> |
| | + | <p> Nowadays, scientists have put great attentions on biofuels, hoping to find a solution to energy crisis and climate change. Among all kinds of biofuels, alkanes stand out because of its excellent properties. In the research of alkane bio-synthesis, there is always a need to sense or detect alkanes. However, due to the inconspicuous property of alkanes, the current alkane detection or sensing methods can hardly meet the demands of alkane sensing or detection.</p> |
| | + | <p> Our project offers a novel solution to the problem. In our project we constructed a sensor, named AlkSensor, that could respond to certain alkanes. AlkSensor is composed of a transcription factor protein ALKR and a inducible promoter PalkM. We developed a mathematical model to find ways to optimize and regulate the sensor. After the construction and reconstruction of AlkSensor, we performed an in vivo alkane sensing test. The qualitative test showed AlkSensor could function as we expected. Besides, we characterized AlkSensor with different inducers and obtained the quantitative relationship between AlkSensor’s input and output. What is more, we designed an selection strategy based on AlkSensor to select out the strains with high productivity of alkanes . A preliminary test was performed and the result verified the feasibility of this design.</p> |
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