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| | <li><a href="/Team:Hong_Kong-CUHK/Project/overview" id="overview">Overview</a></li> | | <li><a href="/Team:Hong_Kong-CUHK/Project/overview" id="overview">Overview</a></li> |
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| - | <li><a href="/Team:Hong_Kong-CUHK/Project/background">Background</a></li> | + | <li><a class="selected" href="/Team:Hong_Kong-CUHK/Project/background">Background</a></li> |
| | <li><a class="list-2" href="/Team:Hong_Kong-CUHK/Project/Halorhodopsin">Halorhodopsin</a></li> | | <li><a class="list-2" href="/Team:Hong_Kong-CUHK/Project/Halorhodopsin">Halorhodopsin</a></li> |
| | <li><a class="list-2" href="/Team:Hong_Kong-CUHK/Project/Chloride Sensing Unit">Chloride Sensing Unit</a></li> | | <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 class="list-2" href="/Team:Hong_Kong-CUHK/Project/Data_page">Data Page</a></li> | | <li><a class="list-2" href="/Team:Hong_Kong-CUHK/Project/Data_page">Data Page</a></li> |
| | <li><a class="list-2" href="/Team:Hong_Kong-CUHK/Project/light">Light Intra-tunable System</a></li> | | <li><a class="list-2" href="/Team:Hong_Kong-CUHK/Project/light">Light Intra-tunable System</a></li> |
| - | <li><a class="list-2 selected" href="/Team:Hong_Kong-CUHK/Project/electricity">Solar Electricity Generation</a></li> | + | <li><a class="list-2" href="/Team:Hong_Kong-CUHK/Project/electricity">Solar Electricity Generation</a></li> |
| | <li><a href="/Team:Hong_Kong-CUHK/Project/further">Future Applications</a></li> | | <li><a href="/Team:Hong_Kong-CUHK/Project/further">Future Applications</a></li> |
| | <li><a href="/Team:Hong_Kong-CUHK/Project/Judging Form">Judging Form</a></li> | | <li><a href="/Team:Hong_Kong-CUHK/Project/Judging Form">Judging Form</a></li> |
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| | <div class="span-17 last" id="electricity"> | | <div class="span-17 last" id="electricity"> |
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| - | <h3>Electricity Generation</h3> | + | <h2>Previous related projects - a review</h2><br/><br/> |
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| - | <p><strong>1. Our entropy-mixing electrodes were successfully fabricated and functioned properly.</strong></p>
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| | <p> | | <p> |
| - | A pair of entropy-mixing electrodes was fabricated according to F. La Mantia<sup>1</sup>, with the help of Department of Electronic Engineering of CUHK (Fig. 1). | + | In 2010 iGEM competition, Queens-Canada team submited <a href="http://en.wikipedia.org/wiki/Halorhodopsin">halorhodopsin</a> from <em>H. salinarum</em> as a biobrick and inserted this gene into <em>C. elegans</em>. However, it was not well characterized. This year, we are trying to clone <a href="http://en.wikipedia.org/wiki/Halorhodopsin">halorhodopsin</a> 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>. |
| | </p> | | </p> |
| - | <img src="http://www.cse.cuhk.edu.hk/~zwang9/igem/img/e1.jpg" />
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| - | <p>Fig. 1 Mixing-entropy electrodes was successfully fabricated.</p>
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| - | <img src="http://www.cse.cuhk.edu.hk/~zwang9/igem/img/e2.jpg" />
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| - | <p>Fig. 2 Pounder X-ray diffraction (XRD) data showing the property of our electrodes matched published data<sup>1</sup>.</p>
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| | + | </p> |
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| - | In order to know whether our electrodes were fabricated properly, we performed pounder X-ray diffraction (XRD) (Fig. 2). Our data match that described by Arnold et. al.<sup>1</sup>. Therefore, it suggests that our electrodes were successfully constructed. | + | In previous iGEM projects, various light sensors have been developed, including red light sensor (UT Austin, 2004) 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 converting solar energy as chemical potential and further turned it into electricity. Our project would provide a wilder scope of applications from signal transduction and gene regulation to energy harvesting. |
| | </p> | | </p> |
| | <p> | | <p> |
| - | <p>In order to determine the performance of our electrodes, we performed the electricity generation cycle illustrated by F. La Mantia<sup>1</sup> (Fig. 3). Our data showed that our electrodes could perform complete electricity generation cycle (Fig. 4). We started the cycle by immersing the electrodes into 50 ml of 0.6 M sodium chloride solution. The voltage kept increasing, which matched the step 1 of F. La Mantia’s data<sup>1</sup>. After 40 minutes, we shifted the solution to 0.01 M of NaCl solution and the voltage dropped immediately. Thereafter we tried to draw current from this device. We alternatively measured the voltage between the electrodes and the current in the complete circuit (Fig. 5). Both voltage and current were detected, indicating the mixing-entropy electrodes worked as we expected. </p>
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| - | <img src="http://www.cse.cuhk.edu.hk/~zwang9/igem/img/e3.png" />
| + | </p> |
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| - | <p>Fig. 3 Experimental setup for electricity generation. </p>
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| | <p> | | <p> |
| - | <img src="http://www.cse.cuhk.edu.hk/~zwang9/igem/img/e4.png" />
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| - | <p>Fig. 4 Electricity generation cycle was successfully performed. Electrodes were first placed in 25 ml of 0.6 M NaCl solution. At the time of dash line, electrodes were transferred to 0.01 M NaCl solution.</p>
| + | </p> |
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| - | <img src="http://www.cse.cuhk.edu.hk/~zwang9/igem/img/e5.png" />
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| - | <p>Fig. 5 Current was successfully drawn from the device.(A) Voltage varies along with time. (B) Current varies along with time. Voltage and current are alternatively measured by one single electricity multimeter during the process.</p>
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| - | <p><strong>2. <a href="http://en.wikipedia.org/wiki/Halorhodopsin">Halorhodopsin</a>-tramsformed <a href="http://en.wikipedia.org/wiki/E._coli">E. coli</a> can successfully generate electricity.</strong></p>
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| | <p> | | <p> |
| - | <p>To prove our concept, we replaced pure NaCl solution with <a href="http://en.wikipedia.org/wiki/Halorhodopsin">Halorhodopsin</a>-transformed bacteria culture with OD600 = 0.4 (Fig. 6). We first exposed the culture under light, whose intensity was 914.2 lux, until the voltage achieved steady. Thereafter we shut down the light and the light intensity was 142.2 lux. We measured the voltage change, following the discharge process of mixing-entropy battery. We recorded and calculated the average rate of potential change in the presence of light and in the absence of light (Fig. 7). The rate of potential change was significantly larger than that before shutting down the light. We measured the current in a complete circuit and the average current was 0.42 0.05 mA with 15 ohm resistor.</p>
| + | References |
| - | | + | </p> |
| - | <p>According to Cottrell equation, the current is proportional to the surface area of electrodes. However, the surface area of each electrode in our experiment is 12 cm<sup>2</sup> and only 25 ml of bacteria culture was used. If we enlarge the surface area of the electrodes, only 2.86 m<sup>2</sup> is sufficient to boost the current to 1 A with a larger volumn of bacteria culture. Furthermore, we could reduce the surface area of the electrodes by using bacteria culture with high bacterial density. More optimization can be made to improve the performance. Our design has great potential to be widely applied in the future.</p>
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| - | <img src="http://www.cse.cuhk.edu.hk/~zwang9/igem/img/e6.png" />
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| - | <p>Fig. 6 Mixing-entropy electricity generation setting with <a href="http://en.wikipedia.org/wiki/Halorhodopsin">Halorhodopsin</a>-transformed bacteria.</p>
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| - | <img src="http://www.cse.cuhk.edu.hk/~zwang9/igem/img/e7.png" />
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| - | <p>Fig. 7 Voltage changed significantly after light was shut down at steady state. Electrodes were immersed in 25 ml of bacteria culture (OD600=0.4) at high light intensity (914.2 lux). The voltage was steady. The light was turn down to 142.2 lux after 30 minutes. The voltage decreased significantly and 0.42 mA current was generated.</p>
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| - | <p><strong>3. Large-scale light-mediated electricity generation</strong></p>
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| - | <img style="max-width: 680px;" src="http://www.cse.cuhk.edu.hk/~zwang9/igem/img/entropy.jpg" />
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| - | <p>Fig. 8 Large-scale design of light-mediated electricity generation plant </p>
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| - | <p>
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| - | <p>Although we can successfully use the mixing-entropy battery to generate electricity, it does have some limitations. First of all, as we use seawater as the source of saline water, the use of this battery is restricted to the regions near the estuaries. Secondly, the efficiency of this battery is constricted by the chloride concentration of seawater which is around 0.5-0.6 M. </p>
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| - | <p>In order to convert solar energy to electricity in a larger scale and extend its application to more districts, we improved this prototype to a light-driven electricity power generation plant, (Fig. 8). We separate the whole process into three parts: salinity separation, power generation and material recycling, aiming at maximizing the efficiency of power generation at each part.</p>
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| - | <p>In salinity separation, there are two reservoirs containing high salinity sodium chloride solution and low salinity sodium chloride solution respectively. They are connected by a pipeline, which allows material exchange and bacteria movement. <a href="http://en.wikipedia.org/wiki/Halorhodopsin">Halorhodopsin</a>-transformed bacteria move uni-directionally from low salinity reservoir towards high salinity reservoir. There is illumination to the low salinity reservoir, the pipeline and the high salinity reservoir. Bacteria in these regions can uptake chloride ion, in order to carry chloride ions from low salinity reservoir to high salinity reservoir against chloride ion diffusion. </p>
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| - | <p>By this means the salinity potential between low salinity reservoir and high salinity reservoir can be maintained. We propose to employ magnetic field to control the movement of bacteria. </p>
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| - | <p>To overcome this obstacle, we proposed an alternative approach for bacteria movement.
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| - | In the nature, a group of bacteria called magnetotatic bacteria, with inborn special organelle magnetosomes, have magnetotaxis to move along magnetic field as weak as the earth’s magnetic field. In 2003, D. Schultheiss et al. successfully developed transformation platform for Magnetospirillum gryphiswaldense, one model organism of magnetotatic bacteria<sup>2</sup>. It is possible to accomplish salinity separation with <a href="http://en.wikipedia.org/wiki/Halorhodopsin">Halorhodopsin</a>-transformed magnetotatic bacteria under the control of magnetic field, even the earth’s magnetic field.</p>
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| - | <p>Similar to the design of mixing-entropy battery, in power generation, high salinity sodium solution and low salinity solution from reservoir respectively are alternatively flushed to power generation compartment cycle by cycle. After power generation, solutions are collected in high salinity collection tank and low salinity collection tank respectively. These two tanks are also connected by a pipeline, which allows bacteria to move from high salinity solution towards low salinity solution while releasing chloride ions in dark. All bacteria, solution and materials are returned back to reservoirs respectively to repeat the recycling process.</p>
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| - | <p><strong>4. Controlled Bacterial Magnetic-movement</strong></p>
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| - | <p>In order to prove the concept of controlled cell movement, we performed this assay. Rapid uni-directional movement of living cells in a controlled manner continuously provides concentration gradient of the solution. The gradient is important for electricity generation. Once we could control the site where bacteria absorb Cl- and the site where bacteria release Cl-, a concentration gradient of Cl- could be maintained which is critical for a constant electricity generation.</p><p>
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| - | The strategy for controlling cell movement is through providing a steady attractive force which directs the cell movement. Micro-magnetic particles which contains anti-E.coli antibody allow E.coli to attach to the particle. Once the bacteria attach to the particle, we can attract the bacteria by a magnetic field. </p><p>
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| - | In our proof-of-concept design, we first mixed and incubated the antibody-tagged micro-magnetic particles and the bacteria with RFP Coding Device (BBa_J04450). After the incubation, bacteria would attach on the particles to form bacterial complexes. The complexes could be isolated by magnetic field. The complexes were then added to a salt solution for illustrating our biobrick BBa_K559010 functioning under NaCl solution. A magnetic field was applied again to remove the bacterial complexes from the solution.</p>
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| - | <img src="http://www.cse.cuhk.edu.hk/~zwang9/igem/img/future.png" />
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| - | <p>Fig. 9. This is the microscopic view of the bacteria with RFP Coding Device (BBa_J04450) coated on antibody-tagged micromagnetic particles</p>
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| - | <p>The results show magnetic field controlled the movement of the bacteria being captured and moved by our control.
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| - | </p><p>In our method, we use 5ug of antibodies tagged on magnetic particles to capture the bacteria. Without magnetic complex capturing bacterial cell, the O.D. 600 after 1 hour incubation did not change much. While, with magnetic complex capturing bacterial cells, the O.D. 600 after 1 hour incubation change around 0.2, that is 4x107 cells/ug antibodies used.</p>
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| - | <img src="http://www.cse.cuhk.edu.hk/~zwang9/igem/img/future1.png" />
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| - | <img src="http://www.cse.cuhk.edu.hk/~zwang9/igem/img/future2.png" />
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| - | <img src="http://www.cse.cuhk.edu.hk/~zwang9/igem/img/future3.png" />
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| - | <img src="http://www.cse.cuhk.edu.hk/~zwang9/igem/img/future4.png" />
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| - | <p>Fig. 10. Under magnetic attraction (magnet was located on the right side), the bacterial complex started to move, shown by the red arrow. </p>
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| - | <p>The below video shows the formation of bacterial-magnetic complex.</p>
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| - | <iframe width="560" height="315" src="http://www.youtube.com/embed/2wDCdNoJKjQ" frameborder="0" allowfullscreen></iframe>
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| - | <p>
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| - | Movie 1: Excess bacteria with Biobrick BBa_J04450 transformed were placed in container. Fixed amount of antibody-tagged micro-magnetic particles were added and incubated for 1 hour at room temperature. The bacterial-magnetic complex were isolated by a fixed magnetic field.</p>
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| - | <iframe width="560" height="315" src="http://www.youtube.com/embed/UkOIdZweKyI" frameborder="0" allowfullscreen></iframe>
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| - | <p>
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| - | Movie 2: The bacterial magnetic complexes were placed in NaCl solution for. After its function, the complexes were removed from the salt solution by a fixed magnetic field applied for 30s. </p>
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| - | <iframe width="560" height="315" src="http://www.youtube.com/embed/Psx9S4Yz9YI" frameborder="0" allowfullscreen></iframe>
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| - | Movie 3: The microscopic view on the movement of bacterial complexes under magnetic field. The micromagnetic particle with bacteria loaded on it showsed a spherical shape. In the presence of a magnetic field, the complexes moved along the magnetic field towards the magnet, and they might conjugate together to form a large bacterial complex.
| + | 1. 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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| - | <p><strong>From proof-of-concept to large scale design</strong></p>
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| - | <p>
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| - | Although we can successfully use the mixing-entropy battery to generate electricity, it does have some limitations. First of all, as we use seawater as the source of saline water, the use of this battery is restricted to the regions near the estuary. Secondly, the efficiency of this battery is constricted by the chloride concentration of seawater which is around 0.5-0.6 M. </p><p>
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| - | In order to convert solar energy to electricity in a larger scale and extend its application to more districts, we improved this prototype to a light-driven electricity power generation plant, (Fig. 8). We separate the whole process into three parts: salinity separation, power generation and material recycling, aiming at maximizing the efficiency of power generation at each part.</p>
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| - | <p>
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| - | In salinity separation, there are two reservoirs containing high salinity sodium chloride solution and low salinity sodium chloride solution respectively. They are connected by a pipeline, which allows material exchange and bacteria movement. <a href="http://en.wikipedia.org/wiki/Halorhodopsin">Halorhodopsin</a>-transformed bacteria move uni-directionally from low salinity reservoir towards high salinity reservoir. There is illumination to the low salinity reservoir, the pipeline and part of the high salinity reservoir. Bacteria in these regions can uptake chloride ion, in order to carry chloride ions from low salinity reservoir to high salinity reservoir against chloride ion diffusion. </p>
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| - | <p>
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| - | By this means the salinity potential between low salinity reservoir and high salinity reservoir can be maintained. We propose to employ magnetic field to control the movement of bacteria. </p><p>
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| - | Although using antibody-tagged micro-magnetic particles to allocate bacteria and control its movement is workable, as shown in our result part, it is not suitable to use in large scale. It is because when antibody tagging is used in a large scale, a large number of antibodies are needed and it will severely increase the construction cost of the system. Also, as the stability of the tagged antibodies is not high, when it is used in such a complex and large system, there is high likelihood that the antibodies will detach from the bacteria and therefore reduce the efficiency of the system.</p>
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| - | <p>To overcome this obstacle, we proposed an alternative approach for bacteria movement.</p><p>
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| - | In the nature, a group of bacteria called magnetotatic bacteria, with inborn special organelle magnetosomes, have magnetotaxis to move along magnetic field as weak as the earth’s magnetic field. In 2003, D. Schultheiss et al. successfully developed transformation platform for Magnetospirillum gryphiswaldense, one model organism of magnetotatic bacteria1. It is possible to accomplish salinity separation with <a href="http://en.wikipedia.org/wiki/Halorhodopsin">Halorhodopsin</a>-transformed magnetotatic bacteria under the control of magnetic field, even the earth’s magnetic field.</p><p>
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| - | Similar to the design of mixing-entropy battery, in power generation, high salinity sodium solution and low salinity solution from reservoir respectively are alternatively flushed to power generation compartment cycle by cycle. After power generation, solutions are collected in high salinity collection tank and low salinity collection tank respectively. These two tanks are also connected by a pipeline, which allows bacteria to move from high salinity solution towards low salinity solution while releasing chloride ions in dark. All bacteria, solution and materials are returned back to reservoirs respectively to repeat the recycling process.</p>
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| - | <p>
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| - | Although using antibody-tagged micro-magnetic particles to allocate bacteria and control its movement is workable, as shown in our result part, it is not suitable to use in large scale. It is because when antibody tagging is used in a large scale, a large number of antibodies are needed and it will severely increase the construction cost of the system. Also, as the stability of the tagged antibodies is not high, when it is used in such a complex and large system, there is high likelihood that the antibodies will detach from the bacteria and therefore reduce the efficiency of the system.</p>
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| | </p> | | </p> |
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| - | References | + | |
| | </p> | | </p> |
| | <p> | | <p> |
| - | 1. La Mantia, F. et al. Batteries for Efficient Energy Extraction from a Water Salinity Difference. <em>Nano letters</em>0-3(2011).at <<a href="http://pubs.acs.org/doi/abs/10.1021/nl200500s>">http://pubs.acs.org/doi/abs/10.1021/nl200500s</a>> | + | |
| - | </p>
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| - | <p>
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| - | 2. Schultheiss, D. & Schüler, D. Development of a genetic system for Magnetospirillum gryphiswaldense. <em>Archives of microbiology</em> <strong>179</strong>,89-94(2003).
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| | </div> | | </div> |
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