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Description

To convert optical energy into electric energy in a clean and sustainable way, Optopia is designed as a photovoltaic system consisting of two subsystems: photosynthetic microorganism system (Synechocystis sp. or Rhodopseudomonas palustris) and electrogenic microorganism system (Shewanella oneidensis). Synthetic biology strategies are applied to the system to trigger production and export of lactate in photosynthetic microorganisms, as well as to improve efficiency of lactate utilization and extracellular electron generation in electrogenic microorganism system.

Synechocystis, one kind of cyanobacteria, is more mature than Rhodopseudomonas in lactate production, but generating lots of oxygen during photosynthesis. Given the fact that Shewanella favors anaerobic environment for electricity production, Rhodopseudomonas may serve as a better carbon resource provider for Shewanella, not only because of its anaerobic photosynthesis maintaining an anaerobic environment required for extracellular electron generation in Shewanella, but also due to its capacity of reusing the waste from Shewanella. Hence, we construct a Synechocystis- Shewanella MFC and a Rhodopseudomonas - Shewanella MFC to find an optimized version of Optopia, maximzing the conversion of optical energy to electric energy.

Part1: Photosynthetic microorganism system

1.1 Synechocystis sp.

Synechocystis sp. PCC 6803 is often chosen as engineering microorganism to convert optical energy into chemical energy. In our project, we chose Synechocystis PCC6803 for optical energy conversion. Synechocystis sp. PCC 6803 produces lactate for the electricity production of Shewanella. However, Synechocystis sp. PCC 6803 itself lacks a pathway for producing lactate, and as a photoautotrophic microorganism, Synechocystis sp. PCC 6803 lacks a lactate transporter to transport lactate out of the cell[1]. Therefore, we have designed two strategies to modify Synechocystis sp. PCC 6803.

Strategy 1:

The lactate dehydrogenase gene and the lactate transporter gene are combined in one circuit to achieve lactate production and transportation. For lactate dehydrogenase gene, we chose ldhD, while ldhDc is a codon-optimized version of ldhD, ldhDnARSdR is ldhD with D176A/I177R/F178S/N180R, and ldhDARSdR is the codon-optimized version of ldhDnARSdR. These codon optimizations are aimed at increasing the production of lactate. The lldP protein gene is used to transport the lactate out of the cell. The lldP protein has 12 transmembrane alpha-helical segments and generally lacks cleaved signal sequences. Besides, it cotransports lactate with a proton[1]. In order to detect the expression of these two genes, we add the Flag and 6 × His sequences respectively after the two.

Figure 1. circuit of ldhD-lldP.

Figure 2. circuit of ldhDC-lldP.

Figure 3. circuit of ldhDnARSdR-lldP.

Figure 4. circuit of ldhDARSdR-lldP.

Strategy 2:

In response to the preference for NADPH in the metabolism of Synechocystis sp. PCC 6803, transhydrogenase gene is used to achieve the goal of producing more NADPH, and the glycerol dehydrogenase gene is used to increase the lactate production. For transhydrogenase gene, we use TH and gldA for glycerol dehydrogenase. The gene lldP is also used to transport the lactate out of the cell. And the same as strategy 1, the Flag and 6 × His sequences are used to detect the expression of the two genes.

Figure 5. circuit of TH-gldA-lldP.

After successfully constructing these gene circuits, the shuttle plasmid pCK306 is used to transform the genes into Synechocystis sp. PCC 6803, and the yellow fluorescent protein gene on the pCK306 plasmid is used to detect the transformation.

1.2 Rhodopseudomonas palustris

Rhodopseudomonas palustris is a kind of Purple Non-sulfur Bacteria. Under anaerobic conditions, Rhodopseudomonas palustris can utilize hydrogen, sodium thiosulfate, hydrogen sulfide as electron donors for photoautotrophic growth. Furthermore, it can also have heterotrophic growth under microaerobic to aerobic conditions [2]. In addition, Rhodopseudomonas palustris is widely used in sewage treatment because it is environmental friendly and accessible [3]. Since it is capable of maintaining an anaerobic environment and reusing the acetate generated by Shewanella, Rhodopseudomonas palustris may be a better carbon source provider for Shewanella compared with Synechocystis sp. PCC 6803. Therefore, we decided to modify Rhodopseudomonas palustris so that it could produce lactate under the anaerobic condition and export it out of the cell. According to KEGG database [4], the metabolic pathways related to lactate metabolism in Rhodopseudomonas palustris are as follows: .

To enhance the production of lactate in Rhodopseudomonas palustris, we plan to promote the conversion efficiency of pyruvate to D-lactate and malate to L-lactate. Therefore, we decide to transfer these two genes, mleS [5] and ldhA, [6] into Rhodopseudomonas palustris to generate more lactate intracelluarly. Considering the necessity of transporting lactate out of the cells, we also apply the gene lldP [7] encoding lactate permease. Since the order of these three genes in a polycistronic lead to different expression levels [8], we build a model to optimize the gene order to maximize the gene expression. Also, we use codon optimized tool Jcat [9] to optimize the codons of these genes.

mleS：malate dehydrogenase, the conversion of malic acid to L-lactate.

ldhA：fermentative D-lactate dehydrogenase, NAD-dependent, convert pyruvate to D-lactate.

lldP：L-lactate permease, the lactate is transported out of the cell.

To validate our modeling results, we continue to build three different gene circuits which can determine the highest lactate production efficiency of our total circuit:

Part2: Electrogenic microorganism system

Shewanella oneidensis

Shewanella oneidensis is becoming more and more popular these years as it could transfer electron to the electrode and generate electricity. This fascinating power mainly attributes to a number of conductive c-type cytochromes (c-Cyts) including OmcA-MtrCAB and CymA. CymA could conduct electrons to the MtrCAB complex. Then, the bacteria would use its extension or vesicles of the outer membrane and periplasm to transfer outer membrane c-type cytochromes.

The amount of electricity produced by Shewanella is closely related to the bacteria’s metabolism.

①. Glycolysis: Glycolysis is the metabolic pathway that converts glucose C6H12O6 into pyruvate. As glyceraldehyde-3-phosphate turns into 1,3-Bisphosphoglycerate, NADH is generated, which would be used to transfer electron.

②. TCA cycle: TCA cycle is a series of chemical reactions happened in mitochondrion. The reactions of the cycle are carried out by eight enzymes and three of them including malate dehydrogenase could help to produce NADH.

③. Pyruvate fermentation: Pyruvate fermentation is a common metabolic pathway in bacteria. Several steps of pyruvate fermentation could also produce NADH and the related enzymes are pyruvate formate-lyase, lactate dehydrogenase and formate dehydrogenase.

Shewanella oneidensis MR-1 prefers to use lactate as its carbon source since the amount of lactate-based biomass is more than acetate-based biomass or pyruvate-based biomass. Dld and lldEFG are D- and L-lactate dehydrogenase enzymes, which is the first step of utilizing lactate. To make the use of lactate more efficiently, we overexpress four genes: dld, lldE, lldF, lldG.[10]

①. dld: dld refers to FAD-dependent D-lactate dehydrogenase which could catalyze D-lactate’s transformation into pyruvate.

②. lldEFG: They could encode a L-lactate dehydrogenase complex which could catalyze D-lactate’s transformation into pyruvate.

To ensure that the genes would be expressed efficiently, we add a promoter before lldEFG:

NADH is a significant part of extracellular electron transfer(EET) as it could carry electron. Strenghthening the regeneration of NADH would make EET more efficiently.

To achieve this goal, we overexpress these four genes: gapA2, mdh, pflB, fdh. [11]

①. gapA: It encodes glyceraldehyde-3-phosphate dehydrogenase which could transform 3- phosphoglyceraldehyde into 1,3- diphosphoglycerate.

②. mdh: It encodes NAD dependent malate dehydrogenase which transforms malate into pyruvate

③. pflB: It encodes pyruvate formate-lyase to transform pyruvate into Acetyl-CoA.

④. fdh: It encodes formate dehydrogenase to transform formate into CO2..

Also, to ensure that the genes would be expressed efficiently, we add an promoter before pflB and fdh:

Part3: Whole design

Design of MFC（Microbial Fuel Cell）

We have designed a bipolar chamber MFC this year. Proton exchange membrane divided it into anode chamber and cathode chamber. Anode chamber containing S.oneidensis, nutrient substance(LB、lactate ) or other electrical producing microbes were sealed to prevent the entry of external oxygen. Considering safety and oxidation-reduction potential, we put ferric chloride solution in cathode chamber so that S.oneidensis can transfer electrons outside of their membranes by electron transport chain. Then electrons will reduce ferric ion into ferrous through carbon cloth and produce electricity. We recorded open circuit voltage curve and load voltage curve of MFCs in each different systems. Also, we have measured the biomass of each system in order to ensure whether the improved electricity could be attributed to more attached Shewanella cells on the anodes or the higher electroactivity of single cell.[11]

Symbiosis

Obviously, the ecological relationship between microorganisms is very complex. There is not only the competition between them for the nutrient, but also the regulation of metabolites among them including induction, transgenosis and synergistic metabolism. Besides, it has been found that the co-culture of microorganisms can improve the electric efficiency of Microbial Fuel Cell under certain conditions.

By consulting literature, we found two kinds of microorganisms——Synechocystis sp. PCC 6803 and Rhodopseudomonas palustris, both of which can utilize light energy and provide lactate to S.oneidensis after doing molecular construction.

In order to provide a basic growth environment, we mix the culture medium of different strains.（Please refer to our protocol section for the composition of the mediums.）

Synechocystis-Shewanella MFC

Lactate produced by Synechocystis PCC6803 can be used as the optimal carbon source for Shewanella. At the same time, oxygen produced by Synechocystis would reduce the efficiency of Shewanella’s electricity production. However, it is proved by our electrogenesis experiment that engineered Synechocystis PCC6803 could function and improve the efficiency.[12].

Rhodopseudomonas-Shewanella MFC

Rhodopseudomonas palustris is another partner we found to produce lactate and help Shewanella to produce electricity. Also, Rhodopseudomonas palustris would not produce oxygen which would reduce the efficiency of electricity production. Besides, acetate which is the metabolic waste of Shewanella could be utilized by Rhodopseudomonas palustris. Thus, we hypothesized that Rhodopseudomonas palustris have more potential as the partner of Shewanella.

Part4: Future plan

4.1 Since the whole circuits of lactate utilization part and NADH production part are separated, we could not improve lactate utilization and NADH production at the same time. We would find a method to integrate these two circuits and get a full-function Shewanella, which has the electrogenesis efficiency to maximum extent.

4.2 As Synechocystis PCC6803 and Rhodopseudomonas palustris are used to transfer light energy to lactate, we would like to replace the promoter with light inducible promoter, which would make the engineered bacteria more intelligent.

4.3 Sewage is a type of waste water which contains many kinds of nutrients. We want to replace LB medium with sewage and make our device much more environmentally friendly.

4.4 We also want to integrate the targeted genes into the genome of engineered bacteria so that we can not only use medium without antibiotics which would reduce the growth rate of bacteria, but also have no need to take the plasmid loss into consideration.

Reference

[1]Henrike Niederholtmeyer, Bernd T. Wolfstädter, David F. Savage, Pamela A. Silver, Jeffrey C. Way. Engineering Synechocystis sp. PCC 6803 to Synthesize and Export Hydrophilic Products. Applied and Environmental Microbiology. 2010, 76(11): 3462-3466.

[2] Research progress and application of Rhodopseudomonas palustris [Journal] Wang Yuexian, Liu Dehai - Henan Animal Husbandry and Veterinary Medicine (Comprehensive Edition) 2015, Issue 1

[3] ZHOU Maohong, ZHAO Xiaowei, WU Xuechang. Study on the ability of photosynthetic bacteria Rhodopseudomonas palustris to assimilate phosphorus[J]. Bulletin of Science and Technology, 2002(02): 142-146.

[4] KEGG, https://www.genome.jp/kegg/pathway.html

[5] Cloning and sequence analysis of the gene encoding Lactococcus lactis malolactic enzyme: relationships with malic enzymes FEMS Microbiol Lett. 1994 Feb 1;116(1):79-86

[6] Fine tuning the transcription of ldhA for d-lactate production August 2012, Volume 39, Issue 8, pp 1209–1217

[7] Transport of L-Lactate, D-Lactate, and Glycolate by the LldP and GlcA Membrane Carriers of Escherichia coli Volume 290, Issue 2, 18 January 2002, Pages 824-829

[8] Enhancement of Hydrogen Production and Carbon Fixation in Purple Nonsulfur Bacterium Bacterium by Synthetic Biology Shou-Chen Lo

[9] Jcat, http://www.jcat.de/#opennewwindow

[10] Genomic reconstruction of Shewanella oneidensis MR-1 metabolism reveals a previously uncharacterized machinery for lactate utilization PNAS February 24, 2009 106 (8) 2874-2879

[11] Modular Engineering Intracellular NADH Regeneration Boosts Extracellular Electron Transfer of Shewanella oneidensis MR‑1 ACS Synth. Biol. 2018, 7, 885−895

[12]Varman A. M., Yu Y., You L. & Tang Y. J. Photoautotrophic production of d-lactate in an engineered cyanobacterium. Microb. Cell Fact. 12, 117 (2013).

College of Life Science & Technology
Huazhong University of Science and Technology
Add: 1037 Luoyu Road, Wuhan, Hubei, China; P.C: 430074
Copyright ©2018 Huazhong University of Science & Technology. Produced By HUST-China

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    font-weight: bold;
 }
+.content{
-.about-text .btn {
+   padding-top:30px;
-  margin-top: 20px;
-  //font-size: 15px;
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    font-weight: 600;
-  text-transform: uppercase;
 }
-#call-to-action-2 p {
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-   color: #5f5f5f;
+   margin: 8px 0;
-  font-size: 15px;
 }
-#call-to-action-2 .btn-primary {
-  border-color: #FFFFFF;
-  /* background: transparent; */
-  margin-top: 50px;
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-blockquote cite a:visited,
-blockquote cite a:visited         { color:#555; }
-/* --- pullquotes --- */
-.pullquote-left {
+th{
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+   text-align:center;
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+   font-size:14px;
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+   font-weight: 800;
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 }
+td
-.pullquote-right {
+{
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+    font-size:14px;
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+    font-weight: 500;
-  font-size:14px;
-  line-height:1.6em;
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 }
+</style>
-/* --- button --- */
+<style type="text/css">
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-.btn-theme {
+   margin: 0 auto; /* optional - if want to center align */
-   color: #fff;
-  background: transparent;
-  border: 1px solid #fff;
-  padding: 12px 30px;
-  font-weight: bold;
 }
-a.btn.btn-details {
+</style>
-    float: right;
+<style>
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-/* --- Heading style --- */
-h4.heading {
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-.heading { margin-bottom: 30px; }
-.heading {
    position: relative;
+   padding: 4px 0;
+  bottom: 27px;
+  background: #eeeeee;
+  display:block
 }
+.jR3DCarouselGalleryCustomeTemplate a{
+   text-decoration: none;
-.widgetheading {
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 }
+</style>
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+<script type="text/javascript" src="https://cdnjs.cloudflare.com/ajax/libs/jquery/1.8.0/jquery-1.8.0.min.js"></script>
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+<script src="https://2018.igem.org/Team:HUST-China/js/jquery?action=raw&amp;ctype=text/javascript"></script>
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+<script src="https://2018.igem.org/Team:HUST-China/js/jR3DCarousel-min?action=raw&amp;ctype=text/javascript"></script>
-  padding-bottom: 9px;
+<!-- <script type="text/javascript" src="src/jR3DCarousel.js"></script> -->
-}
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-  position: relative;
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-/* --- Map --- */
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-}
-.pricing-box-item .pricing-heading h3 strong {
-  font-size:28px;
-  font-weight:700;
-  letter-spacing:-1px;
-  color: #05142b;
-}
-.pricing-box-item .pricing-heading h3 {
-  font-size:32px;
-  font-weight:300;
-  letter-spacing:-1px;
-}
-.pricing-box-item .pricing-terms {
-  text-align: center;
-  background: #fff;
-  display: block;
-  overflow: hidden;
-  padding: 11px 0 5px;
-  /* border-top: 2px solid #BBBBBB; */
-  border-bottom: 2px dotted #CDCDCD;
-}
-.pricing-box-item .pricing-terms  h6 {
-  margin-top: 16px;
-  color: #ff5722;
-  font-size: 22px;
-}
-.pricing-box-item .icon .price-circled {
-    margin: 10px 10px 10px 0;
-    display: inline-block !important;
-    text-align: center !important;
-    color: #fff;
-    width: 68px;
-    height: 68px;
-  padding:12px;
-    font-size: 16px;
-  font-weight:700;
-    line-height: 68px;
-    text-shadow:none;
-    cursor: pointer;
-    background-color: #888;
-    border-radius: 64px;
-    -moz-border-radius: 64px;
-    -webkit-border-radius: 64px;
-}
-.pricing-box-item  .pricing-action{
-  margin:0;
-  text-align:center;
-  padding:30px 0 30px 0;
-}
-.pricing-action .btn{
-  background: #ff5722;
-  color: #fff;
-}
-.menuItem .menu{font-size:20px;font-weight:600;list-style:none;padding:0 0 15px;margin-bottom:20px;display:inline-block;color: #e91e63;}
-.menuItem .menu li{padding: 16px 30px 0px;margin-bottom: 0;display:inline-block;width:100%;float:left;background: #ffffff;line-height: 30px;border-left: 2px solid #e91e63;border-radius: 0;}
-.menuItem .detail{
-font-size:13px;
-color:#444;
-font-weight:normal;
-}
-.menuItem .price{
-font-size:13px;
-float:right;
-font-weight:600;
-}
-/* Testimonials */
-.testimonials blockquote {
-    background: #ffffff none repeat scroll 0 0;
-    border: medium none;
-    color: #666;
-    display: block;
-    font-size: 13px;
-    line-height: 20px;
-    padding: 15px;
-    position: relative;
-}
-.testimonials blockquote::before {
-    width: 0;
-    height: 0;
-  right: 0;
-  bottom: 0;
-  content: " ";
-  display: block;
-  position: absolute;
-    border-bottom: 20px solid #fff;
-  border-right: 0 solid transparent;
-  border-left: 15px solid transparent;
-  border-left-style: inset; /*FF fixes*/
-  border-bottom-style: inset; /*FF fixes*/
-}
-.testimonials blockquote::after {
-    width: 0;
-    height: 0;
-    right: 0;
-    bottom: 0;
-    content: " ";
-    display: block;
-    position: absolute;
-    border-style: solid;
-    border-width: 20px 20px 0 0;
-    border-color: #c5c5c5 transparent transparent transparent;
-}
-.testimonials .carousel-info img {
-    border: 1px solid #cccccc;
-    border-radius: 150px !important;
-    height: 75px;
-    padding: 3px;
-    width: 75px;
-}
-.testimonials .carousel-info {
-    overflow: hidden;
-}
-.testimonials .carousel-info img {
-    margin-right: 15px;
-}
-.testimonials .carousel-info span {
-    display: block;
-}
-.testimonials span.testimonials-name {
-    color: #e91e63;
-    font-size: 16px;
-    font-weight: 300;
-    margin: 23px 0 7px;
-}
-.testimonials span.testimonials-post {
-    color: #656565;
-    font-size: 12px;
-}
-/* ===== Widgets ===== */
-/* --- flickr --- */
-.widget .flickr_badge {
-  width:100%;
-}
-.widget .flickr_badge img { margin: 0 9px 20px 0; }
-footer .widget .flickr_badge {
-    width: 100%;
-}
-footer .widget .flickr_badge img {
-    margin: 0 9px 20px 0;
-}
-.flickr_badge img {
-    width: 50px;
-    height: 50px;
-    float: left;
-  margin: 0 9px 20px 0;
-}
-/* --- Recent post widget --- */
-.recent-post{
-  margin:20px 0 0 0;
-  padding:0;
-  line-height:18px;
-}
-.recent-post h5 a:hover {
-  text-decoration:none;
-}
-.recent-post .text h5 a {
-  color:#353535;
-}
+<script type="text/javascript">
+$(document).ready(function(){
+  var slideImages = [ {src: '../jRCarousel/images/01.jpg'},
+                    {src: '../jRCarousel/images/02.jpg'},
+                    {src: '../jRCarousel/images/03.jpg'},
+                    {src: '../jRCarousel/images/04.jpg'},
+                    {src: '../jRCarousel/images/05.jpg'} ]
+  //var jR3DCarousel;
-footer{
+   jR3DCarousel = $('.jR3DCarouselGallery').jR3DCarousel({
-   padding:50px 0 0 0;
+    width: 550,     /* largest allowed width */
-  color:#f8f8f8;
+    height: 320,    /* largest allowed height */
-}
+    slides: slideImages /* array of images source */
+   });
-footer a {
-  color:#fff;
-}
-footer a:hover {
-  color: #e91e63;
-}
-footer h1, footer h2, footer h3, footer h4, footer h5, footer h6{
-  color: #d3d3d3;
-}
-footer address {
-  line-height:1.6em;
-  color: #797979;
-}
-footer h5 a:hover, footer a:hover {
-  text-decoration:none;
-}
-ul.social-network {
-  list-style:none;
-  margin: 4px 0 0 -25px;
-}
-ul.social-network li {
-  display:inline;
-  margin: 0 5px;
-  /* border: 1px solid #2D2D2D; */
-   padding: 5px 0 0;
-  width: 32px;
-  display: inline-block;
-  text-align: center;
-  height: 32px;
-  vertical-align: baseline;
-}
-#sub-footer{
-  text-shadow:none;
-  color: #dedede;
-  padding:0;
-  padding-top:30px;
-  margin:20px 0 0 0;
-  background: #0a0a0a;
-  border-top: 1px solid #3e3e3e;
-}
-#sub-footer p{
-  margin:0;
-  padding:0;
-}
-#sub-footer span{
-  color: #9e9e9e;
-}
-.copyright {
-  text-align:left;
-  font-size:12px;
-}
-#sub-footer ul.social-network {
-  float:right;
-}
+  var carouselCustomeTemplateProps =  {
-/* scroll to top */
+        width: 550,         /* largest allowed width */
-.scrollup{
+        height: 320,        /* largest allowed height */
-    position:fixed;
+        slideLayout : 'fill',     /* "contain" (fit according to aspect ratio), "fill" (stretches object to fill) and "cover" (overflows box but maintains ratio) */
-    width:32px;
+        animation: 'slide3D',   /* slide | scroll | fade | zoomInSlide | zoomInScroll */
-    height:32px;
+        animationCurve: 'ease',
-    bottom:0px;
+        animationDuration: 1900,
-     right:20px;
+        animationInterval: 2000,
-    background: #e91e63;
+        slideClass: 'jR3DCarouselCustomSlide',
-}
+        autoplay: false,
+        controls: true,     /* control buttons */
-a.scrollup {
+        navigation: ''      /* circles | squares | '' */,
-   outline:0;
+        perspective: 2200,
-  text-align: center;
+        rotationDirection: 'ltr',
-}
+        onSlideShow: slideShownCallback
-a.scrollup:hover,a.scrollup:active,a.scrollup:focus {
+    }
-  opacity:1;
+   function slideShownCallback($slide){
-  text-decoration:none;
+    console.log("Slide shown: ", $slide.find('img').attr('src'))
-}
-a.scrollup i {
-  margin-top: 10px;
-  color: #fff;
-}
-a.scrollup i:hover {
-  text-decoration:none;
-}
-.absolute{
-  position:absolute;
-}
-.relative{
-  position:relative;
-}
-.aligncenter{
-  text-align:center;
-}
-.aligncenter span{
-  margin-left:0;
-}
-.floatright {
-  float:right;
-}
-.floatleft {
-  float:left;
-}
-.floatnone {
-  float:none;
-}
-.aligncenter {
-  text-align:center;
-}
-img.pull-left, .align-left{
-  float:left;
-  margin:0 15px 15px 0;
-}
-.widget img.pull-left {
-  float:left;
-  margin:0 15px 15px 0;
-}
-img.pull-right, .align-right {
-  float:right;
-  margin:0 0 15px 15px;
-}
-article img.pull-left, article .align-left{
-  float:left;
-  margin:5px 15px 15px 0;
-}
-article img.pull-right, article .align-right{
-  float:right;
-  margin:5px 0 15px 15px;
-}
- ============================= */
-.clear-marginbot{
-  margin-bottom:0;
-}
-.marginbot10{
-  margin-bottom:10px;
-}
-.marginbot20{
-  margin-bottom:20px;
-}
-.marginbot30{
-  margin-bottom:30px;
-}
-.marginbot40{
-   margin-bottom:40px;
-}
-.clear-margintop{
-  margin-top:0;
-}
-.margintop10{
-  margin-top:10px;
-}
-.margintop20{
-  margin-top:20px;
-}
-.margintop30{
-  margin-top:30px;
-}
-.margintop40{
-  margin-top:40px;
-}
-.flex-control-paging li a {
-  width: 14px;
-  height: 14px;
-  border-radius: 50%;
-  background: #ffffff;
-  text-indent: 50px;
-  overflow: hidden;
-}
-.btn:hover, .btn:focus {
-  color: #FFF;
-  text-decoration: none;
-  background: #d41a59;
-  border-color: #d41a59;
-}
-/*  Media queries
-============================= */
-@media (min-width: 768px) and (max-width: 979px) {
-  a.detail{
-    background:none;
-    width:100%;
    }
-header  .navbar-nav > li > a{
+   jR3DCarouselCustomeTemplate = $('.jR3DCarouselGalleryCustomeTemplate').jR3DCarousel(carouselCustomeTemplateProps);
-}
-  footer .widget form  input#appendedInputButton {
-      display: block;
-      width: 91%;
-      -webkit-border-radius: 4px 4px 4px 4px;
-       -moz-border-radius: 4px 4px 4px 4px;
-          border-radius: 4px 4px 4px 4px;
-  }
-  footer .widget form  .input-append .btn {
-      display: block;
-      width: 100%;
-      padding-right: 0;
-      padding-left: 0;
-      -webkit-box-sizing: border-box;
-       -moz-box-sizing: border-box;
-          box-sizing: border-box;
-          margin-top:10px;
-  }
-  ul.related-folio li{
+   })
-    width:156px;
+</script>
-    margin:0 20px 0 0;
-   }
-}
-@media (max-width: 767px) {
-.navbar-default .navbar-collapse{border-color: none;/* background: #05142b; */}
-.navbar-default .navbar-toggle .icon-bar {background-color: #868686;border-radius: 0;}
-.navbar-default .navbar-toggle:hover, .navbar-default .navbar-toggle:focus {
-background-color: rgba(221, 221, 221, 0);
-}
-.navbar-default .navbar-collapse, .navbar-default .navbar-form {
-border-color: rgba(255, 255, 255, 0.58);
-margin-top: 15px;
-}
-.flex-caption h3{
-  font-size:28px;
-}
-  body {
-    padding-right: 0;
-    padding-left: 0;
-  }
-  .navbar-brand {
-    margin-top: 18px;
-    border-bottom: none;
-  }
-  .navbar-header {
-    /* margin-top: 20px; */
-    border-bottom: none;
-  }
-  .navbar-nav {
-    border-top: none;
-    float: none;
-    width: 100%;
-  }
-.navbar .nav > .active > a, .navbar .nav > .active > a:hover {background: none;font-weight: 700;color: #10cdff;}
-  header .navbar-nav > li {padding-bottom: 0;padding-top: 0;}
-  header .nav li .dropdown-menu  {
-    margin-top: 0;
-  }
-header  .navbar-nav > li > a{
-  line-height: 20px;
-}
-  .dropdown-menu {
-    position: absolute;
-    top: 0;
-    left: 40px;
-    z-index: 1000;
-    display: none;
-    float: left;
-    min-width: 160px;
-    padding: 5px 0;
-    margin: 2px 0 0;
-    font-size: 13px;
-    list-style: none;
-    background-color: #fff;
-    background-clip: padding-box;
-    border: 1px solid #f5f5f5;
-    border: 1px solid rgba(0, 0, 0, .15);
-    border-radius: 0;
-    -webkit-box-shadow: 0 6px 12px rgba(0, 0, 0, .175);
-        box-shadow: 0 6px 12px rgba(0, 0, 0, .175);
-  }
-  li.active  {
-    border: none;
-    overflow: hidden;
-    /* background: #2e8eda; */
-}
-  .box {
-    border-bottom:1px solid #e9e9e9;
-    padding-bottom:20px;
-  }
-  .flexslider .slide-caption {
-    width: 90%;
-    padding: 2%;
-    position: absolute;
-    left: 0;
-    bottom: -40px;
-  }
-  #inner-headline .breadcrumb {
-    float:left;
-    clear:both;
-    width:100%;
-  }
-  .breadcrumb > li {
-    font-size:13px;
-  }
-  ul.portfolio li article a i.icon-48{
-    width:20px;
-    height:20px;
-    font-size:16px;
-    line-height:20px;
-  }
-  .left-sidebar{
-    border-right:none;
-    padding:0 0 0 0;
-    border-bottom: 1px dotted #e6e6e6;
-    padding-bottom:10px;
-    margin-bottom:40px;
-  }
-  .right-sidebar{
-    margin-top:30px;
-    border-left:none;
-    padding:0 0 0 0;
-  }
-  footer .col-lg-1, footer .col-lg-2, footer .col-lg-3, footer .col-lg-4, footer .col-lg-5, footer .col-lg-6,
-  footer .col-lg-7, footer .col-lg-8, footer .col-lg-9, footer .col-lg-10, footer .col-lg-11, footer .col-lg-12{
-    margin-bottom:20px;
-  }
-  #sub-footer ul.social-network {
-    float:left;
-  }
-  [class*="span"] {
-    margin-bottom:20px;
-  }
-}
-@media (max-width: 480px) {
-  .bottom-article a.pull-right {
-    float:left;
-    margin-top:20px;
-  }
-  .search{
-    float:left;
-  }
-  .flexslider .flex-caption {
-    display:none;
-  }
-  .cta-text {
-    margin:0 auto;
-    text-align:center;
-  }
-  ul.portfolio li article a i{
-    width:20px;
-    height:20px;
-    font-size:14px;
-  }
-}
-.tab-section .col{
-}
-.tab-section .container{
-  padding: 15px;
-  border: 1px solid #eaeaea;
-  background: #fff;
-}
-.active .collapsible-header{color: #e91e63;}
-span.color {
-    color: #e91e63;
-    text-transform: uppercase;
-}
-.input-field label {
-    color: #888;
-  font-size:14px;
-}
-.input-field {
-    position: relative;
-    margin-top: 1rem;
-    float: left;
-    width: 100%;
-}
-.alert-danger {
-    float: left;
-}
-/**********************
-  ****iGEM*************************************************************
- **************/
-<script>
-    if ($('.firstHeading')) $('.firstHeading').remove();
-    </script>
-    <style>
-    html {
-        overflow-x: hidden;
-        width: 100%;
-    }
-    .banner-top {
-        margin-left: 15%;
-    }
-    #igem_home_logo {
-        width: 100%;
-        margin-top: 80px;
-    }
-    @media (min-width: 992px) {
-        .work-content .col-md-4 {
-            width: 25%;
-            margin-right: 8%;
-        }
-    }
-    .carousel-control.left,
-    .carousel-control.right {
-        background-image: none;
-        padding-top: 18%;
-    }
-    .carousel-indicators li {
-        background-color: #fff;
-        border: 1px solid #a94442;
-    }
-    .carousel-indicators li.active {
-        background-color: #f0ad4e;
-    }
-    #contentSub,
-    #contentcontainer,
-    #footer-box,
-    #sitesub,
-    #catlinks,
-    #search-controls,
-    #p-logo,
-    .h3,
-    .printfooter,
-    .firstHeading,
-    .visualClear {
-        display: none;
-        box-sizing: content-box;
-    }
-    #sideMenu,
-    #top_title {
-        display: none;
-        box-sizing: content-box;
-    }
-    #sideMenu .mainMenu_toggle,
-    #sideMenu .mainMenu_Title,
-    #sideMenu .sideMenuTitle,
-    #sideMenu .expand_subMenus {
-        box-sizing: content-box;
-    }
-    #bodyContent h1,
-    #bodyContent h2,
-    #bodyContent h3,
-    #bodyContent h4,
-    #bodyContent h5 {
-        margin-bottom: 0px;
-    }
-    #search_text {
-        color: #000;
-    }
-    #globalWrapper,
-    #content {
-        position: absolute;
-        left: 0;
-        right: 0;
-        top: 0;
-        width: 100%;
-        height: 100%;
-        border: 0px;
-        background-color: transparent;
-        margin: 0px;
-        padding: 0px;
-    }
-    @media (min-width: 1200px) {
-        .container-fluid .container {
-            width: 969px;
-            /* margin-left: 300px; */
-        }
-        .head-container {
-            width: 1170px;
-        }
-    }
-    /* article */
-    .text-content article p {
-        text-indent: 20px;
-        margin: 10px 0;
-        font-size: 15px;
-        line-height: 24px;
-    }
-    .text-content article img {
-        width: 80%;
-        padding-left: 20%;
-        /* margin-left: 20%; */
-    }
-    /*     .mw-content-ltr ol{
-        margin:0;
-    } */
-    ol.carousel-indicators {
-        position: absolute;
-        bottom: 10px;
-        left: 50%;
-        z-index: 15;
-        width: 60%;
-        padding-left: 0;
-        margin-left: -30%;
-        text-align: center;
-        list-style: none;
-    }
-        @charset "utf-8";
-    body {
-        font-family: "Helvetica Neue", Helvetica, Arial, "Microsoft Yahei UI", "Microsoft YaHei", SimHei, "\5B8B\4F53", simsun, sans-serif;
-        color: #666;
-        width:100%;
-    }
-    .logo {
-        padding:0;
-    }
-    #myCarousel {
-        margin: 50px 0 0 0;
-    }
-    #navbar-collapse ul {
-        margin-top:0;
-    }
-    .carousel-inner img {
-        margin: 0 auto;
-    }
-    /*.carousel-control {
-        font-size: 100px;
-    }*/
-    .tab-h1 {
-        width:100%;
-        font-size: 30px;
-        text-align: center;
-        letter-spacing: 1px;
-        color: #ffffff;
-        background-color:#97477c;
-        padding: 30px;
-        margin: 0;
-    }
-    .tab-p {
-        font-size: 20px;
-        text-align: center;
-        letter-spacing: 1px;
-        color: #999;
-        margin: 20px 0 40px 0;
-    }
-    .container-fluid{
-        width:100%;
-    }
-    .tab1 {
-        width:100%;
-        margin: 0 0;
-        color: #666;
-        background:#333;
-    }
-    .tab1 .text-muted {
-        color: #999;
-        text-decoration: line-through;
-    }
-    .tab1 .media-heading {
-        margin: 5px 0 20px 0;
-    }
-    .tab1 .col {
-        padding: 20px;
-    }
-    .text span{
-        color: #97477c;
-    }
-    .text p {
-        font-size: 14px;
-    }
-    #footer {
-        background-color: #eee;
-        border-top: 1px solid #ccc;
-        padding: 20px;
-        text-align: center;
-    }
-    .text h4{
-        padding: 5px 10px;
-    }
-    .text p{
-        padding: 2px 10px;
-    }
-    .text h5{
-         padding: 3px 10px;
-    }
-    .dropdown button{
-        margin: 2px 20px;
-    }
-    /*没有小于768px，是因为Bootstrap3以移动端优先设计*/
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 <body>
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                      <a class="navbar-brand" href="https://2018.igem.org/Team:HUST-China">
-                      <i class="icon-info-blocks material-icons">account_balance</i>
+                       <img style="height: 50px; width:auto;margin-top:-10px; padding:0;" src="https://static.igem.org/mediawiki/2018/c/cb/T--HUST-China--2018-menu-logo-white.png"></a>
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+                    <a class="navbar-brand" href="https://2018.igem.org">
-                       <img style="height: 40px; width:auto; padding:0;margin-top: 20px;" src=" https://static.igem.org/mediawiki/2018/f/f0/T--UC_Davis--igemLogo.jpg"></a>
+                       <img style="height: 60px; width:auto; padding:0;margin-top: -13px;" src=" https://static.igem.org/mediawiki/2018/f/f0/T--UC_Davis--igemLogo.jpg"></a>
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@@ Line 2,267: / Line 148: @@
                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/project/background">Background</a></li>
                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Description">Description</a></li>
+                              <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Applied Design">Applied Design</a></li>
                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Safety">Safety</a></li>
                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Demonstrate">Demonstrate</a></li>
@@ Line 2,276: / Line 158: @@
                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Experiments">Experiments</a></li>
                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Results">Results</a></li>
+                              <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/wetlab/protocols">Protocols</a></li>
                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/InterLab">Interlab</a></li>
                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Notebook">Notebook</a></li>
-                              <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/wetlab/protocols">Protocols</a></li>
                            </ul>
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@@ Line 2,286: / Line 168: @@
                            <ul class="dropdown-menu">
                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Modeling overview">Modeling overview</a></li>
-                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Comparison between PSB">Comparison between PSB</a></li>
+                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/model_of_systems">Model of systems</a></li>
                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Sort of three genes">Sort of three genes</a></li>
-                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Intelligent device software">Intelligent device software</a></li>
+                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Software">Software</a></li>
                            </ul>
                        </li>
@@ Line 2,297: / Line 179: @@
                            <ul class="dropdown-menu">
                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Parts Overview">Parts Overview</a></li>
-                              <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Basic part">Basic part</a></li>
                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Composite part">Composite part</a></li>
                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Part collection">Part collection</a></li>
@@ Line 2,305: / Line 186: @@
                            <a href="#" data-toggle="dropdown" class="dropdown-toggle waves-effect waves-dark">HP<b class="caret"></b></a>
                            <ul class="dropdown-menu">
-                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Collaborations">Human Practices</a></li>
+                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Human Practices">Human Practices</a></li>
-                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Education&Engagement">Education&Engagement</a></li>
+                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Education_Engagement">Education&Engagement</a></li>
                            </ul>
                        </li>
@@ Line 2,312: / Line 193: @@
                            <a href="#" data-toggle="dropdown" class="dropdown-toggle waves-effect waves-dark">TEAM<b class="caret"></b></a>
                            <ul class="dropdown-menu">
-                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Team Members">Team Members</a></li>
+                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Team">Team Members</a></li>
                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Collaborations">Collaborations</a></li>
                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Attributions">Attributions</a></li>
@@ Line 2,328: / Line 209: @@
    </section>
    -->
-  </section>
+<section id="inner-headline">
-    <section class="projects">
+  <div class="container">
-    <div class="container">
      <div class="row">
-       <div class="col-md-12">
+       <div class="col-lg-12">
-         <div class="aligncenter"><h2 class="aligncenter">Discription</h2>
+         <h2 class="pageTitle">Description</h2>
-          </strong>
-        </div>
-        <br/>
        </div>
      </div>
-   <div class="container">
+   </div>
-              <div class="row">
+  </section>
-              </div>
+<section style="margin-top: 10px;">
-   </div>
-          <div class="col-md-12 ">
-           <h2 class="color">Shewanella</h2>
-                  </div>
     <div class="container">
-              <div class="row">
+      <video id="my-video" class="video-js" controls preload="auto" width="100%" height="auto"
-              </div>
+      poster="https://static.igem.org/mediawiki/2018/8/83/T--HUST-China--2018-menu-vedio-poster01.png" data-setup="{}">
-   </div>
+        <source src="https://static.igem.org/mediawiki/2018/7/7a/T--HUST-China--2018-menu-vedio04.webm" type="video/webm">
-    <div class="col-md-12">
+        <source src="https://static.igem.org/mediawiki/2018/4/4e/T--HUST-China--2018-menu-vedio03.mp4" type="video/mp4">
-            <p>In the part of Shewanella, we employ Shewanella oneidensis MR-1 as our chassis and pyydt as the plasmid. Shewanella could utilize the lactate as its carbon source and produce electricity with the help of a multiprotein porin-cytochrome complex on its outer membrane called MtrABC. MtrABC could transfer the electron from Shewanella’s inner membrane to the surrounding. pYYDT is a shuttle plasmid between E.Coli and Shewanella. We constructed this plasmid in E.Coli and transferred it into Shewanella by conjugating these two kinds of bacteria.</p>
+        <p class="vjs-no-js"></p>
-          </div>
+        <p class="vjs-no-js">
-    <div class="container">
+          To view this video please enable JavaScript, and consider upgrading to a web browser that
-              <div class="row">
+          <a href="https://videojs.com/html5-video-support/" target="_blank">supports HTML5 video</a>
-              </div>
+        </p>
-   </div>
+      </video>
-            <div class="col-md-8 col-md-offset-2">
+  </div>
-          <img  class="img-responsive" src="https://static.igem.org/mediawiki/2018/b/b6/T--HUST-China--2018-description-logo1.png" alt="showcase image" >
+</section>
-          </div>
-          <div class="container">
-              <div class="row">
-              </div>
-            </div>
-           <div class="col-md-12" style="text-align:center;">
-            <p>Our design of constructing Shewanella is devided into two parts: improvement of utilization of lactate and increment of the amount of NADH in Shewanella.</p>
-         </div>
-         <div class="container">
+  <section class="content">
-              <div class="row">
+  <div class="container">
+        <div class="row">
-              </div>
+            <div class="col-md-12 content-text">
-            </div>
+                 <div class="about-logo">
+                  <p>
+To convert optical energy into electric energy in a clean and sustainable way, Optopia is designed as a photovoltaic system consisting of two subsystems: photosynthetic microorganism system (Synechocystis sp. or Rhodopseudomonas palustris) and electrogenic microorganism system (Shewanella oneidensis). Synthetic biology strategies are applied to the system to trigger production and export of lactate in photosynthetic microorganisms, as well as to improve efficiency of lactate utilization and extracellular electron generation in electrogenic microorganism system. </p>
+<br/>
+<p>
+Synechocystis, one kind of cyanobacteria, is more mature than Rhodopseudomonas in lactate production, but generating lots of oxygen during photosynthesis. Given the fact that Shewanella favors anaerobic environment for electricity production, Rhodopseudomonas may serve as a better carbon resource provider for Shewanella, not only because of its anaerobic photosynthesis maintaining an anaerobic environment required for extracellular electron generation in Shewanella, but also due to its capacity of reusing the waste from Shewanella. Hence, we construct a Synechocystis- Shewanella MFC and a Rhodopseudomonas - Shewanella MFC to find an optimized version of Optopia, maximzing the conversion of optical energy to electric energy.
-         <div class="col-md-12 col-md-offset-1">
+</p>
+  <div class="row">
-            <p>(1). Improvement of utilization of lactate:
+    </div>
-To make the use of lactate more efficiently, we overexpress four genes: dld-Ⅱ, lldE, lldF, lldG.</p>
-          <div class="container">
-              <div class="row">
-              </div>
-            </div>
-            <p>①. dld-Ⅱ: dld-Ⅱ refers to FAD-dependent D-lactate dehydrogenase which could catalyze D-lactate’s transformation into pyruvate. </p>
-              <div class="row1">
-              </div>
+                  <h3><strong>Part1: <span class="red-content">Photosynthetic microorganism system</span></strong></h3>
-            <p>②. lldE/lldF/lldG: They could encode a L-lactate dehydrogenase complex which could catalyze D-lactate’s transformation into pyruvate.</p>
+                 </div>
-            <p>We constructed them separately in pYYDT and found out if there was any improvement at the amount of electricty produced by our Shewanella.</p>
-          </div>
-             <div class="container">
-              <div class="row">
-              </div>
              </div>
-          <div class="col-md-4 col-md-offset-3">
-         <img  class="img-responsive center-block" src="https://static.igem.org/mediawiki/2018/d/db/T--HUST-China--2018-description-logo1.png.png" alt="showcase image">
          </div>
-         <div class="container">
+         <div class="row">
-              <div class="row">
+            <div class="col-md-12 info-blocks">
+                 <i class="icon-info-blocks material-icons"><img class="img-responsive" src="https://static.igem.org/mediawiki/2018/8/8d/T--HUST-China--2018-lanzao01.png"></i>
+                      <div class="info-blocks-in" style="background-color: #ffffff; border: 1px solid #eeeeee;border-radius:5px;">
+                              <h3><b>1.1 Synechocystis sp.</b></h3>
+                              <p>Synechocystis sp. PCC 6803 is often chosen as engineering microorganism to convert optical energy into chemical energy. In our project, we chose Synechocystis PCC6803 for optical energy conversion. Synechocystis sp. PCC 6803 produces lactate for the electricity production of Shewanella. However, Synechocystis sp. PCC 6803 itself lacks a pathway for producing lactate, and as a photoautotrophic microorganism, Synechocystis sp. PCC 6803 lacks a lactate transporter to transport lactate out of the cell[1]. Therefore, we have designed two strategies to modify Synechocystis sp. PCC 6803.</p>
+                             <div class="row">
+                                </div>
+      <div class="col-md-6 col-md-offset-2" style="text-align: center;">
+                            <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/6/64/T--HUST-China--2018-syndes2-picture.png">
+                        </div>
+                            <div class="col-md-12">
+                              <p>Strategy 1:</p>
+                                <p>The lactate dehydrogenase gene and the lactate transporter gene are combined in one circuit to achieve lactate production and transportation. For lactate dehydrogenase gene, we chose ldhD, while ldhDc is a codon-optimized version of ldhD, ldhDnARSdR is ldhD with D176A/I177R/F178S/N180R, and ldhDARSdR is the codon-optimized version of ldhDnARSdR. These codon optimizations are aimed at increasing the production of lactate. The lldP protein gene is used to transport the lactate out of the cell. The lldP protein has 12 transmembrane alpha-helical segments and generally lacks cleaved signal sequences. Besides, it cotransports lactate with a proton[1]. In order to detect the expression of these two genes, we add the Flag and 6 × His sequences respectively after the two.</p>
+                            </div>
+                            <div class="col-md-6 col-md-offset-3" style="text-align: center;">
+                                    <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/b/be/T--HUST-China--2018-experimental_1-ldhD-lldP.png">
+                                    <p>Figure 1. circuit of ldhD-lldP.</p>
+                                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/c/ce/T--HUST-China--2018-experimental_2-ldhDC-lldP.png">
+                                    <p>Figure 2. circuit of ldhDC-lldP.</p>
+                                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/a/a9/T--HUST-China--2018-experimental_3-ldhDnARSdR-lldP.png">
+                                    <p>Figure 3. circuit of ldhDnARSdR-lldP.</p>
+                                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/b/ba/T--HUST-China--2018-experimental_4-ldhDARSdR-lldP.png">
+                                    <p>Figure 4. circuit of ldhDARSdR-lldP.</p>
+                            </div>
+                          <div class="col-md-12">
+                            <p>Strategy 2:</p>
+                              <p>In response to the preference for NADPH in the metabolism of Synechocystis sp. PCC 6803, transhydrogenase gene is used to achieve the goal of producing more NADPH, and the glycerol dehydrogenase gene is used to increase the lactate production. For transhydrogenase gene, we use TH and gldA for glycerol dehydrogenase. The gene lldP is also used to transport the lactate out of the cell. And the same as strategy 1, the Flag and 6 × His sequences are used to detect the expression of the two genes.</p>
+                          </div>
+                          <div class="col-md-6 col-md-offset-3" style="text-align: center;">
+                          <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/c/c4/T--HUST-China--2018-experimental_5-TH-gldA-lldP.png">
+                                    <p>Figure 5. circuit of TH-gldA-lldP.</p></div>
+                           <div class="col-md-12">
+                              <p>After successfully constructing these gene circuits, the shuttle plasmid pCK306 is used to transform the genes into Synechocystis sp. PCC 6803, and the yellow fluorescent protein gene on the pCK306 plasmid is used to detect the transformation.</p>
+                          </div>
+                      </div>
+                  </div>
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-             <div class="col-md-6 col-md-offset-3">
-          <img  class="img-responsive" src="https://static.igem.org/mediawiki/2018/b/b9/T--HUST-China--2018-description-logo2.png.png" alt="showcase image">
+          <div class="row">
-        </div>
+             <div class="col-md-12 info-blocks">
-        <div class="container">
+                <i class="icon-info-blocks material-icons"><img class="img-responsive" src="https://static.igem.org/mediawiki/2018/6/63/T--HUST-China--2018-zhaozehong01.png"></i>
-              <div class="row">
+                      <div class="info-blocks-in" style="background-color: #ffffff; border: 1px solid #eeeeee;border-radius:5px;">
+                          <div class="col-md-12">
+                              <h3><b>1.2 Rhodopseudomonas palustris<b/></h3>
+                                <p>Rhodopseudomonas palustris is a kind of Purple Non-sulfur Bacteria. Under anaerobic conditions, Rhodopseudomonas palustris can utilize hydrogen, sodium thiosulfate, hydrogen sulfide as electron donors for photoautotrophic growth. Furthermore, it can also have heterotrophic growth under microaerobic to aerobic conditions [2]. In addition, Rhodopseudomonas palustris is widely used in sewage treatment because it is environmental friendly and accessible [3]. Since it is capable of maintaining an anaerobic environment and reusing the acetate generated by Shewanella, Rhodopseudomonas palustris may be a better carbon source provider for Shewanella compared with Synechocystis sp. PCC 6803.
+Therefore, we decided to modify Rhodopseudomonas palustris so that it could produce lactate under the anaerobic condition and export it out of the cell. According to KEGG database [4], the metabolic pathways related to lactate metabolism in Rhodopseudomonas palustris are as follows: .</p></div>
+<div  class="col-md-7 col-md-offset-2" > <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/d/da/T--HUST-China--2018-rhododes-picture.png"></div>
+                          <div class="col-md-12">
+                              <p>To enhance the production of lactate in Rhodopseudomonas palustris, we plan to promote the conversion efficiency of pyruvate to D-lactate and malate to L-lactate. Therefore, we decide to transfer these two genes, mleS [5] and ldhA, [6] into Rhodopseudomonas palustris to generate more lactate intracelluarly. Considering the necessity of transporting lactate out of the cells, we also apply the gene lldP [7] encoding lactate permease.
+Since the order of these three genes in a polycistronic lead to different expression levels [8], we build a model to optimize the gene order to maximize the gene expression. Also, we use codon optimized tool Jcat [9] to optimize the codons of these genes.</p>
+                         </div>
+                         <div  class="col-md-7 col-md-offset-2" > <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/0/00/T--HUST-China--2018-description-picture3.png.png"></div>
+                          <div class="col-md-12">
+                              <p>mleS：malate dehydrogenase, the conversion of malic acid to L-lactate.
+</p>
+<p>ldhA：fermentative D-lactate dehydrogenase, NAD-dependent, convert pyruvate to D-lactate.</p>
+<p>lldP：L-lactate permease, the lactate is transported out of the cell.</p>
+<p>To validate our modeling results, we continue to build three different gene circuits which can determine the highest lactate production efficiency of our total circuit:</p>
+                         </div>
+                         <div  class="col-md-7 col-md-offset-2" > <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/0/0d/T--HUST-China--2018-description-picture4.png.png"></div>
+                    <div  class="col-md-7 col-md-offset-2" > <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/6/63/T--HUST-China--2018-description-picture5.png.png"></div>
+                    <div  class="col-md-7 col-md-offset-2" > <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/8/88/T--HUST-China--2018-description-picture6.png.png"></div>
+                  </div>
                </div>
-            </div>
-         <div class="col-md-6 col-md-offset-2">
-        <p>Then we put these four genes in one circuit to level up the production of electricity.</p>
-      </div>
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-              <div class="row">
-              </div>
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-          <img  class="img-responsive" src="https://static.igem.org/mediawiki/2018/4/4f/T--HUST-China--2018-description-logo3.png.png" alt="showcase image">
-        </div>
-        <div class="container">
-              <div class="row">
-              </div>
-            </div>
-            <div class="col-md-6 col-md-offset-2">
-        <p>(2). Increment of the amount of NADH in Shewanella</p>
-      </div>
-        <div class="container">
-              <div class="row">
-              </div>
-            </div>
-            <div class="col-md-12 col-md-offset-2">
-            <p>To achieve this goal, we overexpress these four genes: gapA2, mdh, pflB, fdh*. We chose them according to our reference.</p>
-            <div class="container">
-              <div class="row">
-              </div>
-            </div>
-            <p>①. gapA2: It encodes glyceraldehyde-3-phosphate dehydrogenase which could transform 3- phosphoglyceraldehyde into 1,3- diphosphoglycerate</p>
-            <p>②. mdh: It encodes NAD dependent malate dehydrogenase which transforms malate into pyruvate</p>
-            <p>③. pflB: It encodes pyruvate formate-lyase to transform pyruvate into Acetyl-CoA</p>
-            <p>④. pflB: It encodes pyruvate formate-lyase to transform pyruvate into Acetyl-CoA</p>
            </div>
-           <div class="container">
+        <div class="row">
-              <div class="row">
+            <div class="col-md-12 content-text">
+                 <div class="about-logo">
-              </div>
+                  <h3><strong>Part2: <span class="red-content">Electrogenic microorganism system</span></strong></h3>
+                </div>
              </div>
-               <div class="col-md-12 col-md-offset-3">
-          <img  class="img-responsive" src="https://static.igem.org/mediawiki/2018/b/b6/T--HUST-China--2018-description-logo4.png.png" alt="showcase image" >
          </div>
-        <div class="container">
+          <div class="row">
-              <div class="row">
+            <div class="col-md-12 info-blocks">
+                 <i class="icon-info-blocks material-icons"><img class="img-responsive" src="https://static.igem.org/mediawiki/2018/0/0c/T--HUST-China--2018-xiwa01.png"></i>
+                      <div class="info-blocks-in" style="background-color: #ffffff; border: 1px solid #eeeeee;border-radius:5px;">
+                        <h3><b>Shewanella oneidensis<b/></h3>
+                        <p>Shewanella oneidensis is becoming more and more popular these years as it could transfer electron to the electrode and generate electricity. This fascinating power mainly attributes to a number of conductive c-type cytochromes (c-Cyts) including OmcA-MtrCAB and CymA. CymA could conduct electrons to the MtrCAB complex. Then, the bacteria would use its extension or vesicles of the outer membrane and periplasm to transfer outer membrane c-type cytochromes.</p>
+                        <div class="col-md-6 col-md-offset-2" style="text-align: center;">
+                            <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/f/f0/T--HUST-China--2018-description-picture7.png.png">
+                        </div>
+                            <div class="col-md-12">
+                                <p>The amount of electricity produced by Shewanella is closely related to the bacteria’s metabolism. </p>
+                                 <p>①. Glycolysis: Glycolysis is the metabolic pathway that converts glucose C6H12O6 into pyruvate. As glyceraldehyde-3-phosphate turns into 1,3-Bisphosphoglycerate, NADH is generated, which would be used to transfer electron. </p>
+                                  <p>②. TCA cycle: TCA cycle is a series of chemical reactions happened in mitochondrion. The reactions of the cycle are carried out by eight enzymes and three of them including malate dehydrogenase could help to produce NADH. </p>
+                                    <p>③. Pyruvate fermentation: Pyruvate fermentation is a common metabolic pathway in bacteria. Several steps of pyruvate fermentation could also produce NADH and the related enzymes are pyruvate formate-lyase, lactate dehydrogenase and formate dehydrogenase.</p>
+                            </div>
+                            <div class="col-md-6 col-md-offset-2" style="text-align: center;">
+                            <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/e/e4/T--HUST-China--2018-shewades2-picture.png">
+                        </div>
+                        <div class="col-md-12">
+                                <p>Shewanella oneidensis MR-1 prefers to use lactate as its carbon source since the amount of lactate-based biomass is more than acetate-based biomass or pyruvate-based biomass. Dld and lldEFG are D- and L-lactate dehydrogenase enzymes, which is the first step of utilizing lactate. To make the use of lactate more efficiently, we overexpress four genes: dld, lldE, lldF, lldG.[10]</p>
+                                 <p>①. dld: dld refers to FAD-dependent D-lactate dehydrogenase which could catalyze D-lactate’s transformation into pyruvate. </p>
+                                  <p>②. lldEFG: They could encode a L-lactate dehydrogenase complex which could catalyze D-lactate’s transformation into pyruvate. </p>
+                                    <p>To ensure that the genes would be expressed efficiently, we add a promoter before lldEFG:</p>
+                            </div>
+                            <div class="col-md-6 col-md-offset-2" style="text-align: center;">
+                            <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/e/e5/T--HUST-China--2018-description-picture9.png.png">
+                        </div>
+                          <div class="col-md-12">
+                                <p>NADH is a significant part of extracellular electron transfer(EET) as it could carry electron. Strenghthening the regeneration of NADH would make EET more efficiently.</p>
+                                 <p>To achieve this goal, we overexpress these four genes: gapA2, mdh, pflB, fdh. [11] </p>
+                                  <p>①. gapA: It encodes glyceraldehyde-3-phosphate dehydrogenase which could transform 3- phosphoglyceraldehyde into 1,3- diphosphoglycerate. </p>
+                                    <p>②. mdh: It encodes NAD dependent malate dehydrogenase which transforms malate into pyruvate</p>
+                                     <p>③. pflB: It encodes pyruvate formate-lyase to transform pyruvate into Acetyl-CoA.</p>
+                                     <p>④. fdh: It encodes formate dehydrogenase to transform formate into CO2..</p>
+                                     <p>Also, to ensure that the genes would be expressed efficiently, we add an promoter before pflB and fdh:</p>
+                                 </div>
+                                 <div class="col-md-6 col-md-offset-2" style="text-align: center;">
+                            <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/a/a0/T--HUST-China--2018-description-picture10.png.png">
+                        </div>
                </div>
              </div>
-             <div class="col-md-12 col-md-offset-2">
-        <p>According to our model, we divided these four genes into two groups: gapA2/mdh and pflB/fdh*. </p>
+           <div class="row">
-      </div>
+             <div class="col-md-12 content-text">
-       <div class="container">
+                <div class="about-logo">
-              <div class="row">
+                  <h3><strong>Part3: <span class="red-content">Whole design</span></strong></h3>
-              </div>
+                 </div>
              </div>
-        <div class="col-md-4 col-md-offset-3">
-          <img  class="img-responsive" src="https://static.igem.org/mediawiki/2018/3/35/T--HUST-China--2018-description-logo5.png.png" alt="showcase image">
          </div>
-        <div class="container">
+          <div class="row">
-              <div class="row">
+            <div class="col-md-12 info-blocks">
+                 <i class="icon-info-blocks material-icons"><img class="img-responsive" src="https://static.igem.org/mediawiki/2018/9/98/T--HUST-China--2018-coin17.png"></i>
+                      <div class="info-blocks-in" style="background-color: #ffffff; border: 1px solid #eeeeee;border-radius:5px;">
+                        <h3><b>Design of MFC（Microbial Fuel Cell）<b/></h3>
+                        <p>We have designed a bipolar chamber MFC this year. Proton exchange membrane divided it into anode chamber and cathode chamber. Anode chamber containing S.oneidensis, nutrient substance(LB、lactate ) or other electrical producing microbes were sealed to prevent the entry of external oxygen. Considering safety and oxidation-reduction potential, we put ferric chloride solution in cathode chamber so that S.oneidensis can transfer electrons outside of their membranes by electron transport chain. Then electrons will reduce ferric ion into ferrous through carbon cloth and produce electricity.
+We recorded open circuit voltage curve and load voltage curve of MFCs in each different systems. Also, we have measured the biomass of each system in order to ensure whether the improved electricity could be attributed to more attached Shewanella cells on the anodes or the higher electroactivity of single cell.[11] </p>
+                        <div class="col-md-6 col-md-offset-2" style="text-align: center;">
+                            <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/2/2c/T--HUST-China--2018-description-pic1.png">
+                        </div>
+                            <div class="col-md-12">
+                               <h3><b>Symbiosis<b/></h3>
+                                <p>Obviously, the ecological relationship between microorganisms is very complex. There is not only the competition between them for the nutrient, but also the regulation of metabolites among them including induction, transgenosis and synergistic metabolism. Besides, it has been found that the co-culture of microorganisms can improve the electric efficiency of Microbial Fuel Cell under certain conditions.  </p>
+                                  <p>By consulting literature, we found two kinds of microorganisms——Synechocystis sp. PCC 6803 and Rhodopseudomonas palustris, both of which can utilize light energy and provide lactate to S.oneidensis after doing molecular construction.  </p>
+                                    <p>In order to provide a basic growth environment, we mix the culture medium of different strains.（Please refer to our protocol section for the composition of the mediums.）</p>
+                            </div>
+                        <div class="col-md-12">
+                                <p><b>Synechocystis-Shewanella MFC <b/></p>
+                                 <p>Lactate produced by Synechocystis PCC6803 can be used as the optimal carbon source for Shewanella. At the same time, oxygen produced by Synechocystis would reduce the efficiency of Shewanella’s electricity production. However, it is proved by our electrogenesis experiment that engineered Synechocystis PCC6803 could function and improve the efficiency.[12]. </p>
+                            <div class="col-md-8" style="text-align: center;">
+                            <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/b/b0/T--HUST-China--2018-result-001.png">
+                            </div>
+                            <div class="col-md-12" style="text-align: center;">
+                            <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/7/71/T--HUST-China--2018-description-logo29.png.gif">
+                        </div>
+                        <div class="col-md-12">
+                                  <p><b>Rhodopseudomonas-Shewanella MFC <b/></p>
+                                    <p>Rhodopseudomonas palustris is another partner we found to produce lactate and help Shewanella to produce electricity. Also, Rhodopseudomonas palustris would not produce oxygen which would reduce the efficiency of electricity production. Besides, acetate which is the metabolic waste of Shewanella could be utilized by Rhodopseudomonas palustris. Thus, we hypothesized that Rhodopseudomonas palustris have more potential as the partner of Shewanella.
+</p>
+</div>
+                            </div>
+                            <div class="col-md-8" style="text-align: center;">
+                            <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/0/07/T--HUST-China--2018-result-002.png">
+                            </div>
+                            <div class="col-md-12" style="text-align: center;">
+                            <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/0/0a/T--HUST-China--2018-description-logo30.png.gif">
+                            </div>
                </div>
              </div>
-            <div class="col-md-4 col-md-offset-3">
+          </div>
-           <img  class="img-responsive" src="https://static.igem.org/mediawiki/2018/4/47/T--HUST-China--2018-description-logo10.png" alt="showcase image">
+      <div class="row">
-        </div>
+           <div class="col-md-12">
-         <div class="container">
-              <div class="row">
+  <h3><strong>Part4: <span class="red-content">Future plan</span></strong></h3>
+          <div class="row">
+            <div class="col-md-12 info-blocks">
+<br/>
+<p>4.1 Since the whole circuits of lactate utilization part and NADH production part are separated, we could not improve lactate utilization and NADH production at the same time. We would find a method to integrate these two circuits and get a full-function Shewanella, which has the electrogenesis efficiency to maximum extent.</p>
+<br/>
+<p>4.2 As Synechocystis PCC6803 and Rhodopseudomonas palustris are used to transfer light energy to lactate, we would like to replace the promoter with light inducible promoter, which would make the engineered bacteria more intelligent.</p>
+<br/>
+<p>4.3 Sewage is a type of waste water which contains many kinds of nutrients. We want to replace LB medium with sewage and make our device much more environmentally friendly.</p>
+<br/>
+<p>4.4 We also want to integrate the targeted genes into the genome of engineered bacteria so that we can not only use medium without antibiotics which would reduce the growth rate of bacteria, but also have no need to take the plasmid loss into consideration.</p>
+</div>
                </div>
              </div>
-             <div class="col-md-12 col-md-offset-2">
-        <p>Finally, we put all these genes into one circuit and found out if the amount of electricity could reach the maximum. </p>
-      </div>
-       <div class="container">
-              <div class="row">
-              </div>
-            </div>
-         <div class="col-md-12 col-md-offset-3">
-         <img  class="img-responsive" src="https://static.igem.org/mediawiki/2018/e/e3/T--HUST-China--2018-description-logo6.png.png" alt="showcase image">
-       </div>
-    </section>
-  <!--<section class="section-padding gray-bg">-->
+            <h4><strong>Reference </strong></h4>
+ <p>[1]Henrike Niederholtmeyer, Bernd T. Wolfstädter, David F. Savage, Pamela A. Silver, Jeffrey C. Way. Engineering Synechocystis sp. PCC 6803 to Synthesize and Export Hydrophilic Products. Applied and Environmental Microbiology. 2010, 76(11): 3462-3466.</p>
+ <p><span>[2]
+Research progress and application of Rhodopseudomonas palustris
+[Journal] Wang Yuexian, Liu Dehai - Henan Animal Husbandry and Veterinary Medicine (Comprehensive Edition) 2015, Issue 1</span></p>
+ <p><span>[3] ZHOU Maohong, ZHAO Xiaowei, WU Xuechang. Study on the ability of photosynthetic bacteria Rhodopseudomonas palustris to assimilate phosphorus[J]. Bulletin of Science and Technology, 2002(02): 142-146.</span></p>
+ <p><span>[4] KEGG, https://www.genome.jp/kegg/pathway.html</span></p>
+ <p><span>[5] Cloning and sequence analysis of the gene encoding Lactococcus lactis malolactic enzyme: relationships with malic enzymes FEMS Microbiol Lett. 1994 Feb 1;116(1):79-86 </span></p>
+ <p><span>[6] Fine tuning the transcription of ldhA for d-lactate production August 2012, Volume 39, Issue 8, pp 1209–1217 </span></p>
+ <p><span>[7] Transport of L-Lactate, D-Lactate, and Glycolate by the LldP and GlcA Membrane Carriers of Escherichia coli Volume 290, Issue 2, 18 January 2002, Pages 824-829 </span></p>
+ <p><span>[8] Enhancement of Hydrogen Production and Carbon Fixation in Purple Nonsulfur Bacterium Bacterium by Synthetic Biology Shou-Chen Lo</span></p>
+  <p><span>[9] Jcat, http://www.jcat.de/#opennewwindow  </span></p>
+ <p><span>[10] Genomic reconstruction of
+Shewanella oneidensis MR-1 metabolism reveals a previously uncharacterized machinery for lactate utilization PNAS February 24, 2009 106 (8) 2874-2879  </span></p>
+ <p><span>[11] Modular Engineering Intracellular NADH Regeneration Boosts Extracellular Electron Transfer of Shewanella oneidensis MR‑1  ACS Synth. Biol. 2018, 7, 885−895</span></p>
+ <p><span>[12]Varman A. M., Yu Y., You L. & Tang Y. J. Photoautotrophic production of d-lactate in an engineered cyanobacterium. Microb. Cell Fact. 12, 117 (2013).  </span></p>
+</div>
+</div>
+    </div>
+</section>
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-              Huazhong University of Science and Technology<br>
-              Add: 1037 Luoyu Road, Wuhan, Hubei, China;<br>
-              P.C: 430074<br>
-            </address>
-            <p>
-              <i class="icon-phone"></i> (86) 027-87792270 <br>
-              <i class="icon-envelope-alt"></i> zhanyi@hust.edu.cn
-            </p>
-          </div>
-        </div>
-      </div>
-      <div class="col-sm-4">
-        <div class="widget">
-          <h5 class="widgetheading">Quick Links</h5>
-          <ul class="link-list">
-            <li><a class="waves-effect waves-dark" href="#">Latest Events</a></li>
-            <li><a class="waves-effect waves-dark" href="#">Terms and conditions</a></li>
-            <li><a class="waves-effect waves-dark" href="#">Privacy policy</a></li>
-            <li><a class="waves-effect waves-dark" href="#">Career</a></li>
-            <li><a class="waves-effect waves-dark" href="#">Contact us</a></li>
-          </ul>
-        </div>
-      </div>
-      <div class="col-sm-4">
-        <div class="widget">
-          <h5 class="widgetheading">Latest posts</h5>
-          <ul class="link-list">
-            <li><a class="waves-effect waves-dark" href="#">Lorem ipsum dolor sit amet, consectetur adipiscing elit.</a></li>
-            <li><a class="waves-effect waves-dark" href="#">Pellentesque et pulvinar enim. Quisque at tempor ligula</a></li>
-            <li><a class="waves-effect waves-dark" href="#">Natus error sit voluptatem accusantium doloremque</a></li>
-          </ul>
-        </div>
-      </div>
-    </div>
-  </div>
--->
      <div class="container" >
        <div class="row">
-           <div class="copyright" style="text-align: center;color: gray; font-size: 14px;">
+           <div class="copyright col-md-12" style="text-align: center;color: gray; font-size: 14px;">
                <span>
                  College of Life Science & Technology<br>
@@ Line 2,571: / Line 524: @@
    </footer>
 </div>
+<a href="#" class="scrollup waves-effect waves-dark"><img class="img-responsive" src="https://static.igem.org/mediawiki/2018/7/79/T--HUST-China--2018-menu-return-top.png"></a>
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+<!--
+<script src="https://2018.igem.org/Team:HUST-China/js/jquery?action=raw&amp;ctype=text/javascript"></script>-->
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 <script src="https://2018.igem.org/Team:HUST-China/js/bootstrap-min?action=raw&amp;ctype=text/javascript"></script>
+<script src="https://stackpath.bootstrapcdn.com/bootstrap/4.1.3/js/bootstrap.min.js"></script>
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@@ Line 2,584: / Line 540: @@
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+<script src="https://2018.igem.org/Team:HUST-China/js/Vendor-jquery-zoomslider?action=raw&amp;ctype=text/javascript"></script>
-<script src="js/modernizr.custom.js"></script>
+<script src="https://2018.igem.org/Team:HUST-China/js/Vendor-jquery-isotope?action=raw&amp;ctype=text/javascript"></script>
--->
+<script src="https://2018.igem.org/Team:HUST-China/js/Vendor-jquery-magnific-popup?action=raw&amp;ctype=text/javascript"></script>
-<script src="js/jquery.zoomslider.min.js"></script>
+<script src="https://2018.igem.org/Team:HUST-China/js/Vendor-animate?action=raw&amp;ctype=text/javascript"></script>
-<script src="js/jquery.isotope.min.js"></script>
+<script src="https://2018.igem.org/Team:HUST-China/js/Vendor-custom?action=raw&amp;ctype=text/javascript"></script>
-<script src="js/jquery.magnific-popup.min.js"></script>
-<script src="js/animate.js"></script>
-<script src="js/custom.js"></script>
 <script type="text/javascript">
 </script>
 </body>
-</html>