Team:Hong Kong-CUHK/Project

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<a href="Team:HongKong-CUHK/Project"><img class="title-img" src="http://www.cse.cuhk.edu.hk/~zwang9/igem/img/project.png" /></a>
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<a href="javascript:void(0)"><img class="title-img" src="http://www.cse.cuhk.edu.hk/~zwang9/igem/img/project.png" /></a>
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<ul class="nav-list" id="project-list">
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<li><a id="overview">Overview</a></li>
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<li><a class="selected" href="/Team:Hong_Kong-CUHK/Project/overview" id="overview">Overview</a></li>
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<li><a id="background">Background</a></li>
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<li><a id="characterization">Characterization</a></li>
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<li><a href="/Team:Hong_Kong-CUHK/Project/background">Background</a></li>
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<li><a id="lightcontrol">Light Control</a></li>
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<li><a class="list-2" href="/Team:Hong_Kong-CUHK/Project/Halorhodopsin">Halorhodopsin</a></li>
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<li><a id="electricity">Electricity Generation</a></li>
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<li><a class="list-2" href="/Team:Hong_Kong-CUHK/Project/Chloride Sensing Unit">Chloride Sensing Unit</a></li>
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<li><a id="future">Future</a></li>
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<li><a class="list-2" href="/Team:Hong_Kong-CUHK/Project/Mixing Entropy Battery">Mixing Entropy Battery</a></li>
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<li><a href="javascript:void(0)">Results</a></li>
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<li><a class="list-2" href="/Team:Hong_Kong-CUHK/Project/Data_page">Data Page</a></li>
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<li><a class="list-2" href="/Team:Hong_Kong-CUHK/Project/light">Light Intra-tunable System</a></li>
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<li><a class="list-2" href="/Team:Hong_Kong-CUHK/Project/electricity">Solar Electricity Generation</a></li>
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<li><a href="/Team:Hong_Kong-CUHK/Project/further">Future Applications</a></li>
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<li><a href="/Team:Hong_Kong-CUHK/Project/Judging Form">Judging Form</a></li>
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<h1>Project Description</h1>
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<h2>Objectives</h2>
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<p class="main-content">Our world is obviously facing several urgent problems in which energy crisis is definitely a major one.
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<h3>Previous related projects</h3>
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Nowadays, there are corresponding solutions to this problem. However, we find that the existing solutions are not satisfactory.
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There are solar panels to convert light energy into electricity, but it usually requires a large infrastructure.
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However, the efficiency of the conversion process is still not good enough.
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In order to improve the situation, our project targets at producing electricity more cheaply, more effectively and in a more portable way.</p>
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<h2>How can we achieve this?</h2>
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<p class="main-content">We employ a light-driven ion pump in our project.
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By expressing the pump into our target bacterial strain, we can control the bacteria to pump in ions from the environment,
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and eventually produce a salinity difference. Also, we are going to manufacture a pair of specific electrodes.
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By making use of the electrodes, the salinity difference as well as our genetically engineered bacteria,
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we can generate electricity from the salinity difference which is produced by our genetically engineered bacteria in the presence of light.
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It is a brand new way to generate electricity.</p>
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What we show in our project is only the tip of thewhole iceberg. More possible applications of halorhodopin could benefit notonly synthetic biology but also the human society. Here we propose several moreadvanced applications of halorhodopsin.
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Halorhodopsin could facilitate light detection andlight-coupled inter-cell signal transduction. Light signal could be convertedto intracellular chloride level signal, which thus induces P<sub>gad</sub>downstream genes. The target genes could be constructed to accelerate thesynthesis pathway of quorum sensing signals, such as N-acyl homoserine lactones(AHL)<sup>1</sup>. As a result,one clone of bacteria could be designed as light detector, amplifying andconverting light signal to quorum signal to cooperate with other clones ofbacteria.
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Moreover, halorhodopsin is not the only channel thatcouples light to pump ions. There are other ion channels from retinylideneprotein family sharing similar sequences and structures but pumping other ionsusing light, such as bacteriorhodopsin pumping proton ions out of cells<sup>2</sup> andchannelrhodopsin non-specifically pumping cations into cells<sup>3</sup>. Combining pHsensitive promoter<sup>4</sup> with bacteriorhodopsinand osmolality sensitive promoters (OmpF, developed by iGEM08_NYMU-Taipei in2008) with channelrhodopsin,&nbsp; &nbsp;the light-coupled expression platform can beextended to more accurate and more complex regulation, which facilitatesbacteria being more programmable by computer.
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Together with channelrhodopsin, halorhodopsin alsoenables water desalination utilizing solar energy. Sea water desalination haslong been attractive and difficult to implement efficiently. <em>E. coli</em> which can express halorhodopsinand channelrhodopsin could be one promising way to solve this problem. It ispossible to drive <em>E. coli</em> to capturevarious ions (mainly sodium ions, potassium ions and chloride ions) in seawater and move these ions away by controlling the movement of <em>E. coli</em>. By this means sea water couldbe desalinated for drinking, irrigation or other daily usage.
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In conclusion, halorhodopsin could fulfill the needof utilizing light as signal or energy resource. We believe that halorhodopsinwould be one of the most interesting tools of synthetic biology in the future.
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References
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In 2010 iGEM competition, Queens-Canada team submited <a href="http://en.wikipedia.org/wiki/Halorhodopsin">Halorhodopsin</a> from <em>H. salinarum</em> as biobricks and inserted this gene into <em>C. elegans</em>. However, it was not well characterized. This year, we are trying to clone halorhdopsin from <em>N. pharaonis,</em> which has already been successfully introduced and proved to perform complete light cycles in <em> <a href="http://en.wikipedia.org/wiki/E._coli">E. coli</a>, </em>to our biobrick system<sup>1</sup>. We aim to characterize the efficiency of this <a href="http://en.wikipedia.org/wiki/Halorhodopsin">Halorhodopsin</a> to be a well-documented biobrick and a useful tool in <em> <a href="http://en.wikipedia.org/wiki/E._coli">E. coli</a> </em>.
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1.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Shapiro, J. a Thinking about bacterialpopulations as multicellular organisms. <em>Annual review of microbiology</em> <strong>52</strong>,81-104(1998).
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2.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Hayashi, S., Tajkhorshid, E. &amp; Schulten, K. Moleculardynamics simulation of bacteriorhodopsin’s photoisomerization using ab initioforces for the excited chromophore. <em>Biophysical journal</em> <strong>85</strong>,1440-9(2003).
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3.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Nagel, G. et al. Channelrhodopsin-2, a directly light-gatedcation-selective membrane channel. <em>Proceedings of the National Academy of Sciences</em><strong>100</strong>, 13940(2003).
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4.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; San, K. et al. An Optimization Study of a pH-InduciblePromoter System for High-Level Recombinant Protein Production in Escherichiacoli. <em>Annals of the New York Academy of Sciences</em> <strong>721</strong>,268–276(1994).  
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In previous iGEM projects, various light sensors have been developed, including red light sensor (UT Austin, 2004), green light sensor (Tokyo-Nokogen, 2009) and blue light sensor (University of Edinburgh, 2010). They are all light-induced fusion transcription factors that trigger gene expression under the control of specific promoters, facilitating simply on/off switch and light-coupled communication. However, our design makes <a href="http://en.wikipedia.org/wiki/Halorhodopsin">Halorhodopsin</a> not only a dynamically tunable light sensor – by coupling with chloride sensitive promoters (e.g. P<sub>gad</sub>), but also an energy converter – by storing solar energy as osmolality potential and further converted it into electricity. Our project would provide a wilder scope of applications from signal transduction and gene regulation to energy generation.
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Halorhodopsin, a light-driven ion pump originated from <em>Halobacterium</em>, utilizeslight to specifically transport chloride ions into cells against osmolality. Since chloride ion is a ubiquitousand essential element in most biological systems,halorhodopsin has fascinated property whereit accumulates light energy, the most abundant energy in the world, asintracellular chloride ion level. However, this attracting ion channel hasn’tbeen well-characterized yet in the past competition. It is of our great interest tocharacterize and utilize the gene in our project, seeking the possibilities tobenefit synthetic biology and the human society.  
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In our project, we successfully integratedhalorhodopsin into functional biobricks. We developed and implemented twoinnovative applications based on the property of halorhodopsin: Light-coupledcomputer-aided expression platform and entropy-mixing electrical powergeneration. The result is interesting and presents us the power and potentialof this fascinating tool. We are glad to introduce our achievements in theproject session.
 
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Besides the ideasproven in our projects, there are much more potential applications to bedeveloped. Anything with linkage of light and chloride ions might beaccomplished by this tool. We believe that halorhodopsin would be an excitingand useful biobrick in the future.
 
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Field: Project
 
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Block: Background
 
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Session: P<sub>gad</sub> introduction
 
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P<sub>gad</sub> is chloride-sensitive promoter whichwas first discovered in <em>Lactococcuslactis</em><sup>1</sup>, whichis a gram-positive bacterium which canlive in acidic environment. P<sub>gad</sub> operon (Fig. 1) provideshydrochloric acid feedback mechanism to adjust intracellular metabolism, inorder to survive in acidic environment<sup>2</sup>. In thisoperon, gadC is glutamate-gamma-aminobutyrate antiporter and gadB is glutamatedecarboxylase. They are both involved in intracellular pH regulation andco-expressed in the same operon under the control of P<sub>gad</sub><sup>2</sup>. The genebefore P<sub>gad</sub>, named gadR, which is constitutively expressed under thecontrol of P<sub>gadR</sub>, is a positive regulator of P<sub>gad</sub> coupledgenes while intracellular chloride is level elevated<sup>2</sup>. Whenintracellular pH decreases, the expression of gadB and gadC is enhanced due tothe action of gadR and confers glutamate-dependent acid resistance in <em>L. lactis</em><sup>2</sup>.
 
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J. Sanders et al. tried to developchloride-sensitive expression cassette using P<sub>gad</sub> operon<sup>3</sup>.&nbsp; They constructed the cassette from bp 821 to2071 of GenBank sequence AF005098, which includes P<sub>gadR</sub>, gadR, P<sub>gad</sub>and the starting codon ATG, and replaced downstream report genes<sup>3</sup>. They managetransforming the cassette to <em>E. coli</em>and varying the expression of report genes under different sodium chlorideconcentrations<sup>3</sup>. In ourproject, we try to build light-coupled chloride expression switch based on thisdesign.
 
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1.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Sanders, J.W. et al. Identifcation of asodium chloride-regulated promoter in Lactococcus lactis by single-copychromosomal fusion with a reporter gene. <em>Mol Gen Genet</em> <strong>257</strong>, 681-685(1998).
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1.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Hohenfeld, I. Purification of histidine tagged bacteriorhodopsin, pharaonis <a href="http://en.wikipedia.org/wiki/Halorhodopsin">Halorhodopsin</a> and pharaonis sensory rhodopsin II functionally expressed in Escherichia coli. <em>FEBS Letters</em> <strong>442</strong>,198-202(1999).  
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2.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Sanders, J.W. et al. A chloride-inducible acid resistancemechanism in Lactococcus lactis and its regulation. <em>Molecular microbiology</em><strong>27</strong>, 299-310(1998).
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3.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Sanders, J.W., Venema, G. &amp; Kok, J. A chloride-induciblegene expression cassette and its use in induced lysis of Lactococcus lactis. <em>Appliedand environmental microbiology</em> <strong>63</strong>, 4877(1997).  
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<h1>Electricity generation</h1>
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<h2>From salinity to electricity</h2>
 
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A novel nano-electrode has been proposed by Fablo. et. el that electrical energy can be generated by alternating salinity difference. With the materials sponsored from companies in mainland China in surprisingly large quantities, we have reproduced a even larger electrode (in surface area) with ease. With this at hand, we can turn salinity difference from bacterial action to electrical energy we can use.
 
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The graph above showed the voltage variation of our cell against time.
 
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From the graph above, it is clear that the voltage generated from the cell culture cannot power up any daily electronic device.
 
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An IC manufactured by Seiko, S-882Z, is a voltage booster that accepts input voltage down to 0.3V. This IC is produced  through fully-depleted Silicon-On-Insulator technology that enables such low voltage input. The output is 1.8V/100uA; this voltage is used to charge up a super-capacitor. The supercapacitor can act as a voltage source for dc-dc converters, provide up to 5V for low-power device applications.
 
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{{Template:Team:Hong_Kong-CUHK/TEMPLATE_2011_FOOT}}

Latest revision as of 02:51, 6 October 2011

Previous related projects

 

In 2010 iGEM competition, Queens-Canada team submited Halorhodopsin from H. salinarum as biobricks and inserted this gene into C. elegans. However, it was not well characterized. This year, we are trying to clone halorhdopsin from N. pharaonis, which has already been successfully introduced and proved to perform complete light cycles in E. coli, to our biobrick system1. We aim to characterize the efficiency of this Halorhodopsin to be a well-documented biobrick and a useful tool in E. coli .

 

In previous iGEM projects, various light sensors have been developed, including red light sensor (UT Austin, 2004), green light sensor (Tokyo-Nokogen, 2009) and blue light sensor (University of Edinburgh, 2010). They are all light-induced fusion transcription factors that trigger gene expression under the control of specific promoters, facilitating simply on/off switch and light-coupled communication. However, our design makes Halorhodopsin not only a dynamically tunable light sensor – by coupling with chloride sensitive promoters (e.g. Pgad), but also an energy converter – by storing solar energy as osmolality potential and further converted it into electricity. Our project would provide a wilder scope of applications from signal transduction and gene regulation to energy generation.

 

 

References

1.        Hohenfeld, I. Purification of histidine tagged bacteriorhodopsin, pharaonis Halorhodopsin and pharaonis sensory rhodopsin II functionally expressed in Escherichia coli. FEBS Letters 442,198-202(1999).

 

 

 


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