Team:Peking/firsttry/killing

From 2014.igem.org

(Difference between revisions)
Line 9: Line 9:
<link rel="stylesheet" type="text/css" href="https://2014.igem.org/Team:Peking/firsttry/css/1001inside.css?action=raw&ctype=text/css" />
<link rel="stylesheet" type="text/css" href="https://2014.igem.org/Team:Peking/firsttry/css/1001inside.css?action=raw&ctype=text/css" />
<link rel="stylesheet" type="text/css" href="https://2014.igem.org/Team:Peking/firsttry/css/1001hide.css?action=raw&ctype=text/css" />
<link rel="stylesheet" type="text/css" href="https://2014.igem.org/Team:Peking/firsttry/css/1001hide.css?action=raw&ctype=text/css" />
-
   
+
 
<script type="text/javascript" src="https://2014.igem.org/Team:Peking/firsttry/js/jquery.js?action=raw&ctype=text/javascript"></script>
<script type="text/javascript" src="https://2014.igem.org/Team:Peking/firsttry/js/jquery.js?action=raw&ctype=text/javascript"></script>
Line 15: Line 15:
<script type="text/javascript" src="https://2014.igem.org/Team:Peking/firsttry/js/inside.js?action=raw&ctype=text/javascript"></script>
<script type="text/javascript" src="https://2014.igem.org/Team:Peking/firsttry/js/inside.js?action=raw&ctype=text/javascript"></script>
<script type="text/javascript" src="https://2014.igem.org/Team:Peking/firsttry/js/members.js?action=raw&ctype=text/javascript"></script>
<script type="text/javascript" src="https://2014.igem.org/Team:Peking/firsttry/js/members.js?action=raw&ctype=text/javascript"></script>
-
+
 
<!-- InstanceEndEditable -->
<!-- InstanceEndEditable -->
</head>
</head>
Line 23: Line 23:
<div id="mainbody">
<div id="mainbody">
<div id="top1">
<div id="top1">
-
  <nav id="topnav">
+
<nav id="topnav">
-
      <ul>
+
<ul>
-
      <li id="logo" > <img src="https://static.igem.org/mediawiki/2014/0/04/Peking_igemlogo.png" width="80" height="65"  alt=""/></li>
+
<li id="logo" > <img src="https://static.igem.org/mediawiki/2014/0/04/Peking_igemlogo.png" width="80" height="65"  alt=""/></li>
-
      <li id="home"> <a class="a1a" href="https://2014.igem.org/Team:Peking/firsttry">Home</a></li>
+
<li id="home"> <a class="a1a" href="https://2014.igem.org/Team:Peking/firsttry">Home</a></li>
-
        <li id="Team"> <a class="a1a" href="#">Team</a>
+
<li id="Team"> <a class="a1a" href="#">Team</a>
-
        <ul class="sublist">
+
<ul class="sublist">
-
            <li id="Teamsublist"> <a class="a2a" href="https://2014.igem.org/Team:Peking/firsttry/menbers">Members</a></li><li id="Teamsublist"> <a class="a2a" href="#">Notebook</a></li> <li id="Teamsublist"> <a class="a2a" href="#">Attributions</a></li>    </ul>
+
<li id="Teamsublist"> <a class="a2a" href="https://2014.igem.org/Team:Peking/firsttry/menbers">Members</a></li><li id="Teamsublist"> <a class="a2a" href="#">Notebook</a></li> <li id="Teamsublist"> <a class="a2a" href="#">Attributions</a></li>    </ul>
-
        </li>
+
</li>
-
        <li id="Project"> <a class="a1a" href="#">Project</a>
+
<li id="Project"> <a class="a1a" href="#">Project</a>
-
        <ul class="sublist">
+
<ul class="sublist">
-
            <li id="Teamsublist"> <a class="a2a" href="#">Killing</a></li><li id="Teamsublist"> <a class="a2a" href="#">Killing Improvements </a></li> <li id="Teamsublist"> <a class="a2a" href="#">Degradation</a></li>      <li id="Teamsublist"> <a class="a2a" href="#">Suicide</a></li>      </ul>
+
<li id="Teamsublist"> <a class="a2a" href="#">Killing</a></li><li id="Teamsublist"> <a class="a2a" href="#">Killing Improvements </a></li> <li id="Teamsublist"> <a class="a2a" href="#">Degradation</a></li>      <li id="Teamsublist"> <a class="a2a" href="#">Suicide</a></li>      </ul>
-
        </li>
+
</li>
-
        <li id="Modeling"> <a class="a1a" href="#">Modeling</a>
+
<li id="Modeling"> <a class="a1a" href="#">Modeling</a>
-
        <ul class="sublist">
+
<ul class="sublist">
-
            <li id="Teamsublist"> <a class="a2a" href="#">Binding Evaluation </a></li> <li id="Teamsublist"> <a class="a2a" href="#">Project Application</a></li>  <li id="Teamsublist"> <a class="a2a" href="#">Cellular Burdern </a></li>      </ul>
+
<li id="Teamsublist"> <a class="a2a" href="#">Binding Evaluation </a></li> <li id="Teamsublist"> <a class="a2a" href="#">Project Application</a></li>  <li id="Teamsublist"> <a class="a2a" href="#">Cellular Burdern </a></li>      </ul>
-
        </li>
+
</li>
-
        <li id="Achievements" style="width:18%"> <a class="a1a" href="#">Achievements</a>
+
<li id="Achievements" style="width:18%"> <a class="a1a" href="#">Achievements</a>
-
        <ul class="sublist">
+
<ul class="sublist">
-
            <li id="Teamsublist"> <a class="a2a" href="#">Parts</a></li>
+
<li id="Teamsublist"> <a class="a2a" href="#">Parts</a></li>
-
            <li id="Teamsublist"> <a class="a2a" href="#">Judging Criteria </a></li>    </ul>
+
<li id="Teamsublist"> <a class="a2a" href="#">Judging Criteria </a></li>    </ul>
-
        </li>
+
</li>
-
        <li id="Safety "> <a class="a1a" href="#">Safety</a></li>
+
<li id="Safety "> <a class="a1a" href="#">Safety</a></li>
-
        <li id="Human" style="width:18%"> <a class="a1a" href="#">Human Practice</a></li>
+
<li id="Human" style="width:18%"> <a class="a1a" href="#">Human Practice</a></li>
-
    </ul>
+
</ul>
-
  </nav>
+
</nav>
</div>
</div>
Line 59: Line 59:
<div id="insidemid">
<div id="insidemid">
-
   
+
 
-
    <aside id="sidenav">
+
<aside id="sidenav">
-
        <div id="list">
+
<div id="list">
-
        <ul id= "fnav">    
+
<ul id= "fnav">
<!-- InstanceBeginEditable name="wcginside1" -->
<!-- InstanceBeginEditable name="wcginside1" -->
<!--*******************导航栏*******************导航栏*******************导航栏*******************导航栏*******************导航栏**********************-->
<!--*******************导航栏*******************导航栏*******************导航栏*******************导航栏*******************导航栏**********************-->
-
        <li><a href="#killing01">Introduction</a></li>
+
<li><a href="#731">A. Water bloom in Taihu Lake</a></li>
-
        <li><a href="#killing02">Design</a></li>
+
<li><a href="#732">Development of water bloom</a></li>
-
        <li><a href="#killing03">Result</a></li>
+
<li><a href="#733">Existing treatments</a></li>
-
       
+
<li><a href="#734">Views</a></li>
 +
<li><a href="#735">Our Project</a></li>
 +
 
<!--*******************导航栏结束*******************导航栏结束*******************导航栏结束*******************导航栏结束**********************-->
<!--*******************导航栏结束*******************导航栏结束*******************导航栏结束*******************导航栏结束**********************-->
<!-- InstanceEndEditable -->
<!-- InstanceEndEditable -->
-
        </ul>
+
</ul>
-
        </div>
+
</div>
-
</aside>
+
</aside>
-
     
+
 
-
    <div id="midbody">
+
<div id="midbody">
<!-- InstanceBeginEditable name="wcginside2" -->
<!-- InstanceBeginEditable name="wcginside2" -->
-
<!--*******************正文*******************正文*******************正文*******************正文*******************正文*******************-->  
+
<!--*******************正文*******************正文*******************正文*******************正文*******************正文*******************-->
-
  <h2 id="killing01">Introduction</h2>
+
<h2 id="731">A. Water bloom in Taihu Lake</h2>
-
      <p> Algal blooms seriously threaten the ecological integrity and sustainability of aquatic ecosystems. They can not only deplete oxygen thus being harmful to the phytoplankton, and also produce a variety of toxic secondary metabolites such as microcystin. Among many kinds of algae that can cause water bloom, <i>Microcystis Aeruginosa</i> accounts for a significant proportion [1]. We developed a new approach to control the population of <i>Microcystis Aeruginosa</i> in the water which can compensate for the lack of other methods. Our engineered <i>E. coli</i>, which can express and secrete hen egg lysozyme and kill <i>Microcystis Aeruginosa</i> efficiently, safely, and controllably with the help of &#945;- hemolysin type I secretion system in <i>E. coli</i>,. Besides, to avoid our <i>E. coli</i> being under the threat of lysozyme, we also add an immunity system. </p>
+
 
-
  <h2 id="killing02">Design</h2>
+
<p>Over 80% fresh water lakes in China are under the threatens of water blooms, including the five biggest ones(Poyang Lake, Dongting Lake, Taihu Lake, Hongze Lake, and Chaohu Lake).Algal blooms burst annually in these lakes, harming ecosystem and industries, affecting citizens’ ordinary lives. Fresh water is the fundamental basis of our life, but now it has to be suffering. </p>
-
      <h3>1.Hen Design</h3>
+
 
-
<p><i>Microcystis Aeruginosa</i> is a species of freshwater cyanobacteria which can form harmful algal blooms (HABs) [1].
+
<p>Besides China, other countries also treat water blooms as a serious concern as the result of its large scale and hazard.For instance, Erie Lake in North America experienced a large scale water bloom in 2011. The area conquered by cyanobacteria once reached nearly 2000 square miles. Residents around were warning due to the high concentration of Microcystin. </p>
-
It almost has the same cell wall components with gram negative bacteria, such as outer membrane, peptidoglycan and inner membrane.
+
 
-
Peptidoglycan, as an important structural component of bacterial cell wall, can provide resistance against turgor pressure [2].
+
 
-
Peptidoglycan can be cleaved by bacterial cell-wall hydrolases (BCWHs), which will lead to the lysis of bacteria. So we put attention to lysozymes, the well-known and best-studied group of BCWHs.</p>
+
<p>This summer, Peking iGEM Team went to NIGLAS (Nanjing Institute of Geography and Limnology of the Chinese Academy of Sciences) in Nanjing and Taihu Limnology Ecology Observatory in Wuxi. We interviewed researchers and citizens, exploring the mechanism of algal bloom occurrenceand existing anti-bloom treatments(discuss2ed below). Further more, we got the first-hand information of social opinion to water blooms. </p>
-
<p>Among the various kinds of lysozymes, we choose to work with hen egg lysozyme. Hen egg lysozyme, also known as lysozyme C (chicken-type), is one of the most widely used lysozyme, which is easily available. Besides, hen egg lysozyme has high antibacterial effect.</p>
+
 
-
<p>We let our <i>E. coli</i> express hen egg lysozyme. Hen egg lysozyme is a kind of 1,4 -&#946;-N- acetylmuramidase which causes the cleaving of the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the bacterial peptidoglycan, thus cause the lysis of bacteria [3]. We amplified the hen egg lysozyme gene that was synthesized from company (Genscript, Nanjing, China), and put this gene into the plasmid pET-21a to test the efficiency of lysozyme being expressed from engineered <i>E. coli</i>. This plasmid was transformed into <i>E. coli</i> BL21(DE3) and defined as strain A.
+
<figure><img src="https://static.igem.org/mediawiki/2014/1/1c/Peking2014Ycy_Asking_question.JPG"/><figcaption>Peking iGEM 2014 member was asking questions about algae clutivation in NIGLAS</figcaption></figure>
-
</p>
+
 
-
<p>
+
 
-
<figure><img src="https://static.igem.org/mediawiki/2014/e/ee/Peking2014ylq_1.png"/><figcaption>Figure 2: HlyA is the C-terminal signal sequence; HlyB and HlyD are the membrane proteins involved in type I secretion; TolC is an outer membrane protein that is essential in the type I secretion pathway together with membrane proteins HlyB and HlyD.</figcaption></figure></p>
+
<p>Taihu Lake lies in the southeast area of China, adjacent Jiangsu Province and Zhejiang Province. As one of the five biggest lakes in China, Taihu Lake covers 2427.8㎞², and has a 393.2 km longshore. Taihu Lake is big but shallow, with an average depth of 1.89m. </p>
-
<h3>2.Secretion</h3>
+
 
-
<p>To achieve our goal of controlling the growth of <i>Microcystis Aeruginosa</i>, our <i>E. coli</i> should have the ability to secrete hen egg lysozyme. Therefore it`s necessary to introduce a secretion system to our <i>E. coli</i>.</p>
+
<figure><img src = "https://static.igem.org/mediawiki/2014/b/b4/Peking2014Ycy_Taihu_map_1.png"/><figcaption>Maps of Taihu basin area</figcaption></figure>
-
<p>To date, five kinds of translocation pathways have been identified in <i>E. coli</i>. These pathways can either deliver proteins from the cytosol to the medium through only one-step process, or via a periplasmic intermediate which need two steps. In order to prevent the contact between lysozyme and peptidoglycan, we utilized a one-step process system, type I secretion system.</p>
+
<figure><img src = "https://static.igem.org/mediawiki/2014/b/bc/Peking2014Ycy_Taihu_map_2.png"/></figure>
-
<p>Type I secretion system, which is also known as ABC transporter, works in a continuous secretion process across both the inner and the outer membrane of gram-negative bacteria. The proteins involved in Type I secretion system form a channel that exports proteins from the cytoplasm to the extracellular environment. </p>
+
 
-
<p>All of the Type I secretion systems, &#945;-hemolysin(H1yA) secretion system is the best characterized, studied and has been widely used. Therefore we choose to work with this system to achieve the secretion of hen egg lysozyme.</p>
+
<p>From 1987, water bloom began to become a serious concern in Taihu Lake due to gradual eutrophication. It bursts year after year in Taihu Lake, and there seems to be no end of it. Up to now, more than 80% of the water body has been heavily eutrophicated. </p>
-
<p>&#945;-hemolysin(HlyA) secretion system contains 4 parts: They are HlyA, HlyB, HlyD and TolC respectively. HlyA is the C-terminal signal sequence of &#945;-hemolysin, which can be recognized by HlyB. HlyB is an ATP-binding cassette. HlyD is a membrane fusion protein, which can be links between the outer and the inner membrane components of the system. And TolC is a specific outer membrane protein, which forms a long channel throughout the outer membrane and the periplasm, largely open towards the extracellular medium.</p>
+
 
-
<p>We got these genes, HlyB (BBa_M1003), HlyD (BBa_M1004), TolC (BBa_M1027) from iGEM part. We did the Gibson Assembling first to put these 3 genes together in the backbone pSB1C3. In the meantime, we constructed another plasmid, pET-21a, which contains the hen egg lysozyme gene, a Glu-Ser linker, and a HlyA signal sequence at the C-terminal of hen egg lysozyme-GS linker. We did the co-transformation, and put these two plasmid that mentioned above in the same <i>E. coli</i> BL21(DH3), which was defined as Stain A.</p>
+
<p> China government has done much to reverse the exacerbation of environment in Taihu basin area. Nevertheless, things always go the other side of man's willing. </p>
-
<p>In order to improve the efficiency of the hen egg lysozyme secretion, we also constructed a plasmid that contains both the lysozyme-linker-hlyA signal sequence and these 3 components. We transformed this plasmid, whose backbone is pET-21a in the the <i>E. coli</i> BL21(DH3), and then defined as Stain B. We induced Stain A and Stain B with IPTG and then tested the effect of the lysozyme secretion and also the killing effect with the secreted lysozyme. You can see the details in the result. </p>
+
 
-
<h3>3. Immunity System</h3>
+
<p>In 1991, China government launched first phase of Taihu Decontamination Project, hundreds of billions yuan was spent afterwards. </p>
-
<p>The function of lysozyme, as we mentioned above, is to provide hydrolysis of peptidoglycan by bacterial cell-wall hydrolases renders bacteria sensitive to lysis. Under the tremendous threat of lysozymes, bacteria in turn evolved mechanisms to avoid bacteriolysis, such as highly specific and potent lysozyme inhibitors production [4].</p>
+
 
-
<p>There are several inhibitors that are specific for the hen egg lysozyme. YkfE is one of them. It is the product of the ORFan gene, and also known as a kind of ivy (inhibitor of vertebrate lysozyme).
+
<p>In 1998, the government approved "Taihu environmental governance". The same year, governments of Jiangsu, Zhejiang, and Shanghai relevant departments, as well as State Council launched a series of water pollution control movements,the largest one within which is the "Focus on Taihu with no tolerance" action in the end of 1998. The so-called "Attained before zero", is that, before the end of 31st Dec 1998, 1035 key polluting enterprises in Taihu basin area must completely achieve emission standards. Although officially announced the "basicallyaccomplish governance objectives", but public doubt never ends. </p>
-
The ykfE`s inhibition of lysozyme occurs via a key-lock type of interaction, without the conformational changes in the lysozyme inhibitor and lysozyme molecules [5].  
+
 
-
</p>
+
<p>Detection results after 2000 showed no signs of water quality improving in Taihu Lake. </p>
-
<p>In our project, we work with the protein ykfE to protect our <i>E. coli</i> effectively against lysozyme while killing <i>Microcystis Aeruginosa</i> with lysozyme.</p>
+
 
-
<p>Our construct contains the ykfE gene under control of T7 promoter in the pET-21a plasmid was designated Stain C. This pET-21a plasmid was transformed into E. coli BL21, and the resulting strain was designated as ykfE overexpression strain.</p>
+
<p>In 2005, the second phase of Taihu Decontamination Project was started. In a interview of Huang Weixuan, the former vice director of Bureau of Taihu Lake Basin, he mentioned that only 1% water body was polluted in early 80s last century, but more than 80% water body was polluted at that time. </p>
-
<p>We tested the role of ykfE in protection of E. coli against lysozyme. Lysozyme was added in both the overexpression stain and the control stain.</p>
+
 
-
  <h2 id="killing03">Result</h2>
+
<p>After the burst of cyanobacteria in 2007, Wuxi, Jiangsu Province government spent 2 billion yuan as a special fund on Taihu governance. From 2007 to 2011, 2.8 million tons of cyanobacteria was collected from Taihu Lake. </p>
-
<h3>1. The killing efficiency of purified Hen egg lysozyme against algae</h3>
+
 
-
<h4>1.1 Growth Curve of algae</h4>
+
<p>May 2008, a plan was raised by regional governments, aiming to improve Taihu water quality from classV to class IV before 2020, with an expecting cost of 111.5 billion yuan. </p>
-
<p>Two strains of <i>Microcystis Aeruginosa</i> were involved in our experiment: one was FACHB (Freshwater Algae Culture Collection at the Institute of Hydrobiology) 1343 and the other one is Pcc(Pasteur culture collection)-7806.</p>
+
 
-
<p>In this experiment, we measured the OD670nm, the absobance of Chlorophyll a, as a reflection of algal density. OD was monitored every day until the growth of algae reached a plateau.</p>
+
<p>1st November 2011, Li Guoying, the vice minister of the Ministry of Water Resources, stated that Taihu faced difficulties of today and tomorrow, not only contamination but also shortage of water. </p>
-
<h4>1.2. The killing curve of hen egg lysozyme against algae.</h4>
+
 
-
<p> 5 hours after lysozyme solution was added into well-distributed algal culture, the aggregation and sedimentation of algae could be observed, as shown in fig 2(A). Different volume of 1mg/ml lysozyme solution was added into algae to reach a graded final concentration. OD 670 was checked every day until it stopped decreasing. Result is shown in fig 2.(B) .</p>
+
<p>Statistic from decades shows the need of fresh water from Taihu basin is 37 billion m³ per year, while Taihu can only supply 17.7 billion m³. The gap is obvious. </p>
-
<h3>2. The killing efficiency of purified hen egg lysozyme against <i>E. coli</i></h3>
+
 
-
<p>Since the effect of lysozyme on algae is due to its ability to hydrolyze the polysaccharides as indicated in(wenxian) ,The same graded concentration of hen egg lysozyme as described above was added into <i>E. coli</i>, and the absorbance of cell culture at OD 600 to test the sensibility of <i>E. coli</i> to lysozyme..</p>
+
 
-
<h3>3. ykfE confers resistence against lysozyme to <i>E. coli</i> </h3>
+
<p>Wuxi water crisis is a typical case that water bloom affects citizens’ daily lives: </p>
-
<p>Plasmid contains the gene ykfE was transformed into <i>E. coli</i>. Hen egg lysozyme was added into <i>E. coli</i> Stain C to a final concentration of *** and the absorbance at OD 600 was measured. Result is shown in Fig 5.</p>
+
 
-
<p>Based on the previously mentioned experiment, immunity system was introduced into the engineered E. coli. Similar quantitative experiment was completed and result indicated that ykfE can protect E. coli effectively in the environment of hen egg lysozyme. (Fig. 5)</p>
+
<p>June, 2007, due to continuous south wind and high temperature, a huge amount of cyanobacteria accumulated around the north lakeshore of Meiliang Gulf, where near the intake of waterworks in Wuxi, causing lack of fresh water for 2 million citizens for a week. </p>
-
<h3>4. The killing efficiency of Lysozyme being expressed from engineered <i>E. coli</i></h3>
+
 
-
<p><i>E. coli</i> BL21(DE3) carrying the plasmid pET 21a-lysozyme and <i>E. coli</i> carrying the blank plasmid pET21a as a control were induced with IPTG at the same time using the same protocols as described before. After being broken up and centrifuged, the supernatant was isolated and was analyzed by polyacrylamide gel electrophoresis to verify the existence of lysozyme (Fig 6).</p>
+
<p>In the end of May, temperature in Wuxi reached a historic level. Government reminded citizens doing heatstroke prevention via television and newspaper. However, the real emergency was ignored. 28th, May, many citizen claimed that water flowing from their taps smelly, and received the reply saying that there is nothing wrong with it. One day later, almost all citizens in Wuxi lost fresh water supply. Bottled water sold out within hours, even twice more expensive than usual. Many parents chose milk instead of water as their children’s drinking. </p>
-
<p>Result that presents here shows hen egg lysozyme positive in supernatant of <i>E. coli</i> equipped with the plasmid pET 21a-lysozyme, compared to control group. Both supernatant was then added into <i>Microcystis Aeruginosa</i>, and the killing efficiency can be seen obviously here (Fig 7). </p>
+
 
-
<p>We thus could claim that the lysozyme expressed from our engineered <i>E. coli</i> has the ability to kill <i>Microcystis Aeruginosa</i>.</p>
+
<p>To our surprise, the government didn’t cut off water supply, neither nor warning citizen the potential danger. </p>
-
<h3>5. The killing efficiency of Lysozyme being secreted from engineered <i>E. coli</i> </h3>
+
 
-
<p> <i>E. coli</i> BL21(DE3) carrying the plasmid pET 21a-lysozyme-lard as well as pET 21a-ABC transporter was induced with IPTG. <i>E. coli</i> carrying plasmid pET 21a-lysozyme-lard, pET 21a-ABC transporter and pET 21a-inhibitor, <i>E. coli</i> with plasmid pET 21a-lysozyme-lard only as well as well as <i>E. coli</i> with pET 21a-blank were induced with IPTG as control groups at the same time. After being centrifuged, supernatant was isolated and was analyzed by polyacrylamide gel electrophoresis to verify the existence of lysozyme (Fig 8). </p>
+
<p>A week after the cyanobacteria burst, monitoring data showed another burst in Meiliang gulf. Chlorophyll a concentration overreached 40µg /L, some area over 170 µg /L. Eliminating water bloom in Taihu seems to be beyond man’s power. </p>
-
<p>Result presents here shows Lysozyme positive in supernatant of induced <i>E. coli</i> with pET 21a-lysozyme-lard, pET 21a-ABC transporter and pET 21a-inhibitor and induced <i>E. coli</i> with pET 21a-lysozyme-lard and pET 21a-ABC transporter, while negative in supernatant of <i>E. coli</i> with pET 21a-lysozyme-lard only, or <i>E. coli</i> with pET 21a-blank.</p>
+
 
-
<p>All supernatant was then added into <i>Microcystis Aeruginosa</i>, and the killing effeciency can be seen here (Fig 9). The decrease of OD760nm of culture with supernatant of induced <i>E. coli</i> with pET 21a-lysozyme-lard, pET 21a-ABC transporter and pET 21a-inhibitor as well as induced <i>E. coli</i> with pET 21a-lysozyme-lard and pET 21a-ABC transporter, is in accordance with PAGE result, showing our engineered <i>E. coli</i> are able to kill algae. </p>
+
<p>According statistics, water bloom occurs in Taihu Lake every summer since 1987. After 2000,aggravating of eutrophication and climate changing lead to thatalgal bloomsgradually become a serious environment issue more than an eyesore. </p>
 +
 
 +
<p>Now, Taihu Lake, as well as many other fresh lakes all over the world, is still suffering from water bloom. </p>
 +
 
 +
<p><h2 id="732">B. Development of water bloom </h2></p>
 +
 
 +
<p>Professor Kong Fanxiang in NIGLAS (Nanjing Institute of Geography and Limnology of the Chinese Academy of Sciences) and his fellows raised the four-phase development hypothesis on the process of the cyanobacteria bloom-forming, and they pointed out the burst of cyanobacteria isn’t a sudden event but a result of long-term accumulation. </p>
 +
 
 +
 
 +
<figure><img src = "https://static.igem.org/mediawiki/2014/f/fd/Peking2014Ycy_Professor_kong_report.JPG"/><figcaption>Prof.Kong was giving report to Peking iGEM 2014 Members</figcaption></figure>
 +
 
 +
 
 +
 
 +
 
 +
<p>22ndJuly, Prof. KongFanxiang gave a report on algal bloom to Peking iGEM 2014. </p>
 +
 
 +
<p>During the report, Prof. Kong introduced the overview of lakes in China, lake eutrophication in China, interactions between cyanobacteria and lake eutrophication. The most important part is that the four-phase development hypothesis on the process of the cyanobacteria bloom-forming. </p>
 +
 
 +
<p>Presently there are more than 24800 lakes in China, 2800 within which span more than 1 km². Unfortunately, four-fifths of them are in eutrophication state, including almost all lakes in North China area and middle/lower reaches of Yangtze River. </p>
 +
 
 +
<p>Algal bloom is the result of a variation of vertical position of large volume of algal biomass that is gradually developed and accumulated for long time. According to the ecological theory and the in situ observation of thewater bloom inTaihuLake,Prof.Kong and his fellows raised the four-phase development hypothesis on the process of the cyanobacteria bloom-forming, which divided life span of cyanobacteria mainly by meteorological conditions:dormancy in winter, recruitment in spring, growthand float to the water surface in summer and sink to the sediment in autumn. </p>
 +
 
 +
<p>There is different factors influence the growth state of cyanobacteria ineach phase. In dormancy phase, low temperature and illumination limit the growing of algae resulting in dormancy. Though not fully understand the mechanism, factors can be determined as following: low temperature and illumination in dormancy phase; temperature and dissolve oxygen recruitment phase; nutrient growth and float phase; and temperature sink phase. </p>
 +
 
 +
<p>We could learn that water bloom is largely influenced by meteorological condition, especially temperature. Unfortunately, controlling temperature in Taihu scale is impossible for today’s technology. We must find other ways to regulate growth of cyanobacteria. <sup>[1]</sup></p>
 +
 
 +
<p><h2 id="733">C. Existing treatments </h2></p>
 +
 
 +
<p>People do have several methods to fight against water bloom, and they can be divided into physical, chemical, and biological roughly. </p>
 +
 
 +
 
 +
<p><h3>Physical methods:</h3></p>
 +
 
 +
<p>The easiest method, also the most common one, is mechanical salvage. This method hardly has side effects on environment. However, it is inefficient while money and labor wasting. Furthermore, toxins such as microcystin dissolved in water threaten workers’ health. </p>
 +
 
 +
 
 +
<figure><img src = "https://static.igem.org/mediawiki/2014/6/60/Peking2014Ycy_Salvage_2.JPG"/><figcaption>Salvage workers were fighting against water bloom</figcaption></figure>
 +
 
 +
 
 +
 
 +
 
 +
<p>There are some other physical methods like ultraviolet (UV) radiation, ultrasonic cracking. But they all cost too much money.</p>
 +
 
 +
<p>Clay flocculation is another physics method controls harmful algal blooms. There are several successful examples in Japan and Korea. </p>
 +
<figure><img src = "https://static.igem.org/mediawiki/2014/8/80/Peking2014Ycy_Clay_flocculation_1.JPG"/></figure>
 +
<figure><img src = "https://static.igem.org/mediawiki/2014/e/e7/Peking2014Ycy_Clay_flocculation_2.JPG"/><figcaption>Researchers in NIGLAS were doing clay flocculation experiments in Meiliang Gulf, Taihu</figcaption></figure>
 +
 
 +
 
 +
<p>In a 1996 report, workers in South Korea dispersed approximately 60,000 tons of dry yellow loess (a kaolinite-bearing sediment) by barges over 260 k㎡at a loading rate of 400 g/㎡. Removal rates of Cochlodinium polykrikoides were calculated at 90% to 99% up to 2 m depth, with virtually no reported mortality in the caged fish due to clay treatment. </p>
 +
 
 +
<p>A remarkable weakness of this method is that clay cannot kill cyanobacteria, which remains the possibility of refloating of them, especially for big but shallow lakes like Taihu Lake(average depth 1.89m) in China and Victoria Nyanza in Africa. </p>
 +
 
 +
<p><h3>Chemical methods: </h3></p>
 +
 
 +
<p>Tin- and chlorine- containing toxic compounds(biocides) could damage Microcystis.aeruginosacolonies. These toxic increased the influx of potassium and decrease the uptake of sodium, which is necessary to keep the metabolism of cyanobacteria, especially maintaining intracellular pH. But because of the potential harm to other organisms in lakes, chemical methods can only be taken in some special occasions, a small close fishpond for example. </p>
 +
 
 +
<p>Now, most chemical methods are used in waterworks treating water taken from natural resources to eliminate organisms and chemical compounds. Strong oxidation can kill almost all organisms in water, including cyanobacteria consequently, while degrading chemical compounds into small harmless molecules. </p>
 +
 
 +
<p>Another assay which was widely used is launching copper sulfate or other copper compounds<sup>[2]</sup
 +
>(Donald M. Anderson, 2009). Copper can replace magnesium in Chlorophyll, blocking photosynthesis in cyanobacteria. However, copper can also harm other organisms’ metabolism while inhibiting water blooms. Therefore, these compounds are hardly used now. </p>
 +
 
 +
 
 +
<p><h3>Biological methods: </h3></p>
 +
<p>By introducing competitors(like emerged plants) or predators(like algicidal bacteria) seems to be a ideal method. </p>
 +
 
 +
<p>However,“The common shortcoming in most experiments on preventing microcystis growth is the lack of preliminary calculations and an analysis of the results of long-term monitoring of hydrobiological, hydrochemical, and hydrophysical characteristics of water. Mathematical modeling is seldom used, although it is important for predicting the results of action on microcystis growth. Complex ecological technologies that imply a combined use of inexpensive physicochemical and bio- chemical techniques that do not affect ecological norms have almost completely been ignored.”<sup>[3]</sup>(V.I.Kolmakov Microbiology, 2006) </p>
 +
 
 +
<p>To avoid disadvantages of existing methods, we carefully designed a transgenic E.coli and did a lot of modeling analysis. The detailed introduction can be found on the project pages. </p>
 +
 
 +
<p><h2 id="734">D. Views</h2></p>
 +
 
 +
<p>Taihu Lake basin area, with a population of 49 million people, is one of the most developed, energetic areas in China. Urbanization rate of Taihu Lake basin area is over 70%, whilewhichof China is less than 50%. GDP in this area was 2864.8 billionyuan in 2013. </p>
 +
 
 +
<p>Researchers feel more disappointed when facing the fact that hundreds of billion yuan has been spent but nothing was improved in Taihu Lake, even worsen. </p>
 +
 
 +
<p>In a 2007’s report, Liu Guangzhao, member of World Water Association, and Liu Zhili, professor involved in algae research in Nanjing University, shared the view that pollution in Taihu lake could be eliminated within 5 years. Honestly speaking, there is no obstacle technically while the main obstacle is from the management system. </p>
 +
 
 +
<p>This report listed 5 serious shortages in Taihu Lake governance </p>
 +
 
 +
<p>Technically speaking, pollution in Taihu could be controlled within several years. However, management system inhibits the decontamination. </p>
 +
 
 +
<p>Only 1/10 of spent money is enough for the decontamination once the problems in management were solved. </p>
 +
 
 +
<p>The “863” plan was operated by the interest-related. They made decisions on which project is to be invested, who is to be the project leader, and how effective the projects are. Those projects that really work cannot get enough research funds.</p>
 +
 
 +
<p>Using salvage, instead of other advanced approaches, as the main method in cyanobacteria treatment. </p>
 +
 
 +
<p>Unprecedented is the concerns from states leaders, the enormous cost, and the ineffectiveness.</p>
 +
 
 +
<p>Taihu basin area accounts for only 0.4% of Chinese land area, but with 4% of the country's population, creating 13% of the GDP. This imbalance is bound to cause the contradiction between the river basin development and ecology protection. Management mechanism of chaos intensifies the grim situation of pollution in Taihu. 7 years pasts, what is the actual situation in Taihu? </p>
 +
 
 +
<p>Up to now, eutrophication in Taihu Lake has been eased. Water quality in 15 rivers inflowing into Taihu Lake is above class 4(which means industrially useable), comparing 9 rivers’ water quality is worse than class 5(which mean agriculturally useable) in 2007. </p>
 +
 
 +
<p>According Zhang Min, deputy director of the office of Taihu in Jiangsu Province, eutrophication in Taihu is a long-term outcome and cannot be changes in a relative short time. Marginal  benefits reduced greatly in recent years, which leads to greater difficulties in improving Taihu Lake water quality. Notably, heavy industry is still a big proportion of industrial structure of Taihu Basin Area, and the total discharge of pollutants still exceeds the environmental capacity. </p>
 +
 
 +
<p><h2 id="735">E. Our Project</h2></p>
 +
 
 +
<p>During the interview of researchers in NIGLAS and Taihu, we presented our project and recieved many suggestions. </p>
 +
 
 +
<p>"Ranger amongst enemies" uses molecular assays to eliminate large scale water bloom. Comparing traditional methods, our project provides a whole new view of dealing with water bloom. We endow our rangers weapons, improvement equipments, and a suicide system. Synthetic biology was used to engineered desired organisms. </p>
 +
 
 +
<p>Researchers gave high praise to our project, commenting it can potentially replace methods today. The most noticeable point of our project is that it gives a new way of thinking on water bloom goverance. Though immature, we believe our project open another door leading to the balance between man and nature. </p>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<P>[1] Kong, F., Fao, G. (2005). Hypothesis on cyanobacteria bloom-forming mechanism in large shallow eutrophic lakes. Acta ecologica sinica/Shengtai Xuebao, 25(3), 589-595.</p>
 +
 
 +
<P>[2] Anderson, D. M. (2009). Approaches to monitoring, control and management of harmful algal blooms (HABs). Ocean  coastal management, 52(7), 342-347.</P>
 +
 
 +
<P>[3] Kolmakov, V. I. (2005). [Methods for prevention of mass development of the cyanobacterium Microcystis aeruginosa Kutz emend. Elenk. in aquatic ecosystems]. Mikrobiologiia, 75(2), 149-153.</P>
<!--*******************正文结束*******************正文结束*******************正文结束*******************正文结束*******************正文结束*******************-->
<!--*******************正文结束*******************正文结束*******************正文结束*******************正文结束*******************正文结束*******************-->
Line 143: Line 257:
</body>
</body>
-
<script type="text/javascript">
+
<script type="text/javascript">
$(document).ready(function(){
$(document).ready(function(){
-
$("#sidenav").css( "height", $("#midbody").height()
+
$("#sidenav").css( "height", $("#midbody").height()
-
);
+
);
-
+
 
-
$("#list").pin({
+
$("#list").pin({
-
  containerSelector: "#sidenav"
+
containerSelector: "#sidenav"
-
});
+
});
-
 
+
 
});
});
</script>
</script>
<!-- InstanceEnd --></html>
<!-- InstanceEnd --></html>

Revision as of 16:42, 2 October 2014

A. Water bloom in Taihu Lake

Over 80% fresh water lakes in China are under the threatens of water blooms, including the five biggest ones(Poyang Lake, Dongting Lake, Taihu Lake, Hongze Lake, and Chaohu Lake).Algal blooms burst annually in these lakes, harming ecosystem and industries, affecting citizens’ ordinary lives. Fresh water is the fundamental basis of our life, but now it has to be suffering.

Besides China, other countries also treat water blooms as a serious concern as the result of its large scale and hazard.For instance, Erie Lake in North America experienced a large scale water bloom in 2011. The area conquered by cyanobacteria once reached nearly 2000 square miles. Residents around were warning due to the high concentration of Microcystin.

This summer, Peking iGEM Team went to NIGLAS (Nanjing Institute of Geography and Limnology of the Chinese Academy of Sciences) in Nanjing and Taihu Limnology Ecology Observatory in Wuxi. We interviewed researchers and citizens, exploring the mechanism of algal bloom occurrenceand existing anti-bloom treatments(discuss2ed below). Further more, we got the first-hand information of social opinion to water blooms.

Peking iGEM 2014 member was asking questions about algae clutivation in NIGLAS

Taihu Lake lies in the southeast area of China, adjacent Jiangsu Province and Zhejiang Province. As one of the five biggest lakes in China, Taihu Lake covers 2427.8㎞², and has a 393.2 km longshore. Taihu Lake is big but shallow, with an average depth of 1.89m.

Maps of Taihu basin area

From 1987, water bloom began to become a serious concern in Taihu Lake due to gradual eutrophication. It bursts year after year in Taihu Lake, and there seems to be no end of it. Up to now, more than 80% of the water body has been heavily eutrophicated.

China government has done much to reverse the exacerbation of environment in Taihu basin area. Nevertheless, things always go the other side of man's willing.

In 1991, China government launched first phase of Taihu Decontamination Project, hundreds of billions yuan was spent afterwards.

In 1998, the government approved "Taihu environmental governance". The same year, governments of Jiangsu, Zhejiang, and Shanghai relevant departments, as well as State Council launched a series of water pollution control movements,the largest one within which is the "Focus on Taihu with no tolerance" action in the end of 1998. The so-called "Attained before zero", is that, before the end of 31st Dec 1998, 1035 key polluting enterprises in Taihu basin area must completely achieve emission standards. Although officially announced the "basicallyaccomplish governance objectives", but public doubt never ends.

Detection results after 2000 showed no signs of water quality improving in Taihu Lake.

In 2005, the second phase of Taihu Decontamination Project was started. In a interview of Huang Weixuan, the former vice director of Bureau of Taihu Lake Basin, he mentioned that only 1% water body was polluted in early 80s last century, but more than 80% water body was polluted at that time.

After the burst of cyanobacteria in 2007, Wuxi, Jiangsu Province government spent 2 billion yuan as a special fund on Taihu governance. From 2007 to 2011, 2.8 million tons of cyanobacteria was collected from Taihu Lake.

May 2008, a plan was raised by regional governments, aiming to improve Taihu water quality from classV to class IV before 2020, with an expecting cost of 111.5 billion yuan.

1st November 2011, Li Guoying, the vice minister of the Ministry of Water Resources, stated that Taihu faced difficulties of today and tomorrow, not only contamination but also shortage of water.

Statistic from decades shows the need of fresh water from Taihu basin is 37 billion m³ per year, while Taihu can only supply 17.7 billion m³. The gap is obvious.

Wuxi water crisis is a typical case that water bloom affects citizens’ daily lives:

June, 2007, due to continuous south wind and high temperature, a huge amount of cyanobacteria accumulated around the north lakeshore of Meiliang Gulf, where near the intake of waterworks in Wuxi, causing lack of fresh water for 2 million citizens for a week.

In the end of May, temperature in Wuxi reached a historic level. Government reminded citizens doing heatstroke prevention via television and newspaper. However, the real emergency was ignored. 28th, May, many citizen claimed that water flowing from their taps smelly, and received the reply saying that there is nothing wrong with it. One day later, almost all citizens in Wuxi lost fresh water supply. Bottled water sold out within hours, even twice more expensive than usual. Many parents chose milk instead of water as their children’s drinking.

To our surprise, the government didn’t cut off water supply, neither nor warning citizen the potential danger.

A week after the cyanobacteria burst, monitoring data showed another burst in Meiliang gulf. Chlorophyll a concentration overreached 40µg /L, some area over 170 µg /L. Eliminating water bloom in Taihu seems to be beyond man’s power.

According statistics, water bloom occurs in Taihu Lake every summer since 1987. After 2000,aggravating of eutrophication and climate changing lead to thatalgal bloomsgradually become a serious environment issue more than an eyesore.

Now, Taihu Lake, as well as many other fresh lakes all over the world, is still suffering from water bloom.

B. Development of water bloom

Professor Kong Fanxiang in NIGLAS (Nanjing Institute of Geography and Limnology of the Chinese Academy of Sciences) and his fellows raised the four-phase development hypothesis on the process of the cyanobacteria bloom-forming, and they pointed out the burst of cyanobacteria isn’t a sudden event but a result of long-term accumulation.

Prof.Kong was giving report to Peking iGEM 2014 Members

22ndJuly, Prof. KongFanxiang gave a report on algal bloom to Peking iGEM 2014.

During the report, Prof. Kong introduced the overview of lakes in China, lake eutrophication in China, interactions between cyanobacteria and lake eutrophication. The most important part is that the four-phase development hypothesis on the process of the cyanobacteria bloom-forming.

Presently there are more than 24800 lakes in China, 2800 within which span more than 1 km². Unfortunately, four-fifths of them are in eutrophication state, including almost all lakes in North China area and middle/lower reaches of Yangtze River.

Algal bloom is the result of a variation of vertical position of large volume of algal biomass that is gradually developed and accumulated for long time. According to the ecological theory and the in situ observation of thewater bloom inTaihuLake,Prof.Kong and his fellows raised the four-phase development hypothesis on the process of the cyanobacteria bloom-forming, which divided life span of cyanobacteria mainly by meteorological conditions:dormancy in winter, recruitment in spring, growthand float to the water surface in summer and sink to the sediment in autumn.

There is different factors influence the growth state of cyanobacteria ineach phase. In dormancy phase, low temperature and illumination limit the growing of algae resulting in dormancy. Though not fully understand the mechanism, factors can be determined as following: low temperature and illumination in dormancy phase; temperature and dissolve oxygen recruitment phase; nutrient growth and float phase; and temperature sink phase.

We could learn that water bloom is largely influenced by meteorological condition, especially temperature. Unfortunately, controlling temperature in Taihu scale is impossible for today’s technology. We must find other ways to regulate growth of cyanobacteria. [1]

C. Existing treatments

People do have several methods to fight against water bloom, and they can be divided into physical, chemical, and biological roughly.

Physical methods:

The easiest method, also the most common one, is mechanical salvage. This method hardly has side effects on environment. However, it is inefficient while money and labor wasting. Furthermore, toxins such as microcystin dissolved in water threaten workers’ health.

Salvage workers were fighting against water bloom

There are some other physical methods like ultraviolet (UV) radiation, ultrasonic cracking. But they all cost too much money.

Clay flocculation is another physics method controls harmful algal blooms. There are several successful examples in Japan and Korea.

Researchers in NIGLAS were doing clay flocculation experiments in Meiliang Gulf, Taihu

In a 1996 report, workers in South Korea dispersed approximately 60,000 tons of dry yellow loess (a kaolinite-bearing sediment) by barges over 260 k㎡at a loading rate of 400 g/㎡. Removal rates of Cochlodinium polykrikoides were calculated at 90% to 99% up to 2 m depth, with virtually no reported mortality in the caged fish due to clay treatment.

A remarkable weakness of this method is that clay cannot kill cyanobacteria, which remains the possibility of refloating of them, especially for big but shallow lakes like Taihu Lake(average depth 1.89m) in China and Victoria Nyanza in Africa.

Chemical methods:

Tin- and chlorine- containing toxic compounds(biocides) could damage Microcystis.aeruginosacolonies. These toxic increased the influx of potassium and decrease the uptake of sodium, which is necessary to keep the metabolism of cyanobacteria, especially maintaining intracellular pH. But because of the potential harm to other organisms in lakes, chemical methods can only be taken in some special occasions, a small close fishpond for example.

Now, most chemical methods are used in waterworks treating water taken from natural resources to eliminate organisms and chemical compounds. Strong oxidation can kill almost all organisms in water, including cyanobacteria consequently, while degrading chemical compounds into small harmless molecules.

Another assay which was widely used is launching copper sulfate or other copper compounds[2](Donald M. Anderson, 2009). Copper can replace magnesium in Chlorophyll, blocking photosynthesis in cyanobacteria. However, copper can also harm other organisms’ metabolism while inhibiting water blooms. Therefore, these compounds are hardly used now.

Biological methods:

By introducing competitors(like emerged plants) or predators(like algicidal bacteria) seems to be a ideal method.

However,“The common shortcoming in most experiments on preventing microcystis growth is the lack of preliminary calculations and an analysis of the results of long-term monitoring of hydrobiological, hydrochemical, and hydrophysical characteristics of water. Mathematical modeling is seldom used, although it is important for predicting the results of action on microcystis growth. Complex ecological technologies that imply a combined use of inexpensive physicochemical and bio- chemical techniques that do not affect ecological norms have almost completely been ignored.”[3](V.I.Kolmakov Microbiology, 2006)

To avoid disadvantages of existing methods, we carefully designed a transgenic E.coli and did a lot of modeling analysis. The detailed introduction can be found on the project pages.

D. Views

Taihu Lake basin area, with a population of 49 million people, is one of the most developed, energetic areas in China. Urbanization rate of Taihu Lake basin area is over 70%, whilewhichof China is less than 50%. GDP in this area was 2864.8 billionyuan in 2013.

Researchers feel more disappointed when facing the fact that hundreds of billion yuan has been spent but nothing was improved in Taihu Lake, even worsen.

In a 2007’s report, Liu Guangzhao, member of World Water Association, and Liu Zhili, professor involved in algae research in Nanjing University, shared the view that pollution in Taihu lake could be eliminated within 5 years. Honestly speaking, there is no obstacle technically while the main obstacle is from the management system.

This report listed 5 serious shortages in Taihu Lake governance

Technically speaking, pollution in Taihu could be controlled within several years. However, management system inhibits the decontamination.

Only 1/10 of spent money is enough for the decontamination once the problems in management were solved.

The “863” plan was operated by the interest-related. They made decisions on which project is to be invested, who is to be the project leader, and how effective the projects are. Those projects that really work cannot get enough research funds.

Using salvage, instead of other advanced approaches, as the main method in cyanobacteria treatment.

Unprecedented is the concerns from states leaders, the enormous cost, and the ineffectiveness.

Taihu basin area accounts for only 0.4% of Chinese land area, but with 4% of the country's population, creating 13% of the GDP. This imbalance is bound to cause the contradiction between the river basin development and ecology protection. Management mechanism of chaos intensifies the grim situation of pollution in Taihu. 7 years pasts, what is the actual situation in Taihu?

Up to now, eutrophication in Taihu Lake has been eased. Water quality in 15 rivers inflowing into Taihu Lake is above class 4(which means industrially useable), comparing 9 rivers’ water quality is worse than class 5(which mean agriculturally useable) in 2007.

According Zhang Min, deputy director of the office of Taihu in Jiangsu Province, eutrophication in Taihu is a long-term outcome and cannot be changes in a relative short time. Marginal benefits reduced greatly in recent years, which leads to greater difficulties in improving Taihu Lake water quality. Notably, heavy industry is still a big proportion of industrial structure of Taihu Basin Area, and the total discharge of pollutants still exceeds the environmental capacity.

E. Our Project

During the interview of researchers in NIGLAS and Taihu, we presented our project and recieved many suggestions.

"Ranger amongst enemies" uses molecular assays to eliminate large scale water bloom. Comparing traditional methods, our project provides a whole new view of dealing with water bloom. We endow our rangers weapons, improvement equipments, and a suicide system. Synthetic biology was used to engineered desired organisms.

Researchers gave high praise to our project, commenting it can potentially replace methods today. The most noticeable point of our project is that it gives a new way of thinking on water bloom goverance. Though immature, we believe our project open another door leading to the balance between man and nature.

[1] Kong, F., Fao, G. (2005). Hypothesis on cyanobacteria bloom-forming mechanism in large shallow eutrophic lakes. Acta ecologica sinica/Shengtai Xuebao, 25(3), 589-595.

[2] Anderson, D. M. (2009). Approaches to monitoring, control and management of harmful algal blooms (HABs). Ocean coastal management, 52(7), 342-347.

[3] Kolmakov, V. I. (2005). [Methods for prevention of mass development of the cyanobacterium Microcystis aeruginosa Kutz emend. Elenk. in aquatic ecosystems]. Mikrobiologiia, 75(2), 149-153.