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| | <body> | | <body> |
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| + | <article> |
| − | <section class="header"> | + | |
| − | <div align = "centre">
| + | |
| − | <!-- Navigation -->
| + | |
| − | <header> <a href=""><h4 class="logo">TRINITY</h4></a>
| + | |
| − | <nav>
| + | |
| − | <ul>
| + | |
| − | <li id="links"><a href="">JUDGING FORM</a></li>
| + | |
| − | <li id="links"><a href="">AWARDS</a></li>
| + | |
| − | <li id="links"><a href="">HUMAN PRACTICES </a></li>
| + | |
| − | <li id="links"><a href="">SAFETY</a></li>
| + | |
| − | <li id="links"><a href="">PARTS</a></li>
| + | |
| − | <li id="links"><a href="">PROJECT</a></li>
| + | |
| − | <li id="links"><a href="">TEAM</a></li>
| + | |
| − | </ul>
| + | |
| − | </nav>
| + | |
| − | </header>
| + | |
| − | </div>
| + | |
| − | </section>
| + | |
| − |
| + | |
| − | <section class="main">
| + | |
| − | <div class = "textbox">
| + | |
| − | <h2>Project Description</h2>
| + | |
| − | <h4 class="subheader">The idea</h4>
| + | |
| − | <p class="infobox">
| + | |
| − | Scientists all over the world in different fields are using
| + | |
| − | microorganisms for their research - that is nothing new. But most
| + | |
| − | of them are using monocultures and therefore miss thousands of
| + | |
| − | possibilities which arise in co-cultures. It is estimated that
| + | |
| − | less than 1% of all microorganisms have been successfully
| + | |
| − | cultivated(1). A reason for that might be the very artificial
| + | |
| − | conditions in the laboratory. In nature no organism is completely
| + | |
| − | isolated, but rather lives in very complex systems. Scientist
| + | |
| − | have only just begun to investigate the complex interaction
| + | |
| − | between different microorganisms but yet often fail at the
| + | |
| − | cultivation. Easily obtainable and stable co-cultures would allow
| + | |
| − | research of yet uninvestigated species and might give further
| + | |
| − | insight into cell-cell-interactions between microorganisms(2).
| + | |
| − | One problem of isolated cultures is the regular use of
| + | |
| − | antibiotics as selective pressure and prevention of
| + | |
| − | contaminations which leads towards multiple resistances. Here as
| + | |
| − | well a co-culture with nutrient dependencies instead of higher
| + | |
| − | antibiotic concentration is a better alternative.
| + | |
| − | <br>
| + | |
| − | Pharmacotherapeutic companies spend a lot of money on developing
| + | |
| − | new antibiotics, while in co-culturing conditions some
| + | |
| − | microorganisms show differential gene expression and might
| + | |
| − | produce new antibiotics on their own(3). In general, co-cultures
| + | |
| − | might provide a great opportunity as a new method for cultivation
| + | |
| − | and the production of new antibiotics or industrially interesting
| + | |
| − | products. There is yet a lot of potential for new co-cultures and
| + | |
| − | we - Team iGEM 2018 HHU - want to tackle that challenge.
| + | |
| − | </p>
| + | |
| − | <h4 class="subheader">The challenges</h4>
| + | |
| − | <p class="infobox">
| + | |
| − | Before starting research, first the safety must be ensured. But
| + | |
| − | this is a self solving problem, since the organisms are only able
| + | |
| − | to live in a community that is designed by the researcher itself.
| + | |
| − | That includes a detailed control of the microorganisms, the cell
| + | |
| − | density, their behavior and environmental conditions. For
| + | |
| − | industrial usage the production must be scalable and high
| + | |
| − | throughput must be guaranteed as well. Furthermore, general
| + | |
| − | technologies for a normed use for research projects that are also
| + | |
| − | easily applicable for large scale production must be achieved(4).
| + | |
| − | One goal would be the reduction and simplification of processing
| + | |
| − | steps and easy-to-use protocols for stable co-cultures to make
| + | |
| − | cultivation and production faster and cheaper. Other requirements
| + | |
| − | for making co-cultures a profitable alternative to isolated
| + | |
| − | culturing are big product diversity and efficient extraction of
| + | |
| − | them. For scientists the possibility to build complex systems and
| + | |
| − | methods for big data collection are desirable and highly demanded.
| + | |
| − | </p>
| + | |
| − | <h4 class="subheader">The project</h4>
| + | |
| − | <p class="infobox">
| + | |
| − | Since our co-cultures should be universally usable for
| + | |
| − | laboratories all over the world, we plan to design it as easy and
| + | |
| − | modular as possible. Therefore, the Golden Gate modular cloning
| + | |
| − | standard techniques (MoClo) were used besides Gibson Assembly for
| + | |
| − | our cloning steps, which allows exchange of all parts to adjust
| + | |
| − | the co-culture to every possible condition(5)(6).
| + | |
| − | </p>
| + | |
| − | <p class="infobox">
| + | |
| − | We envision a standardised system, a three-way co-culture with a
| + | |
| − | selection of organisms and regulations. `Trinity` is going to
| + | |
| − | incorporate different dependencies between the organisms in order
| + | |
| − | to control their growth and enable them to adjust each others
| + | |
| − | behaviour.
| + | |
| − | <br>
| + | |
| − | Project Trinity is divided into three systems.
| + | |
| − | <br>
| + | |
| − | Our system 1 deals with the dependence of the organisms
| + | |
| − | <i>Escherichia coli</i>, <i>Saccharomyces cerevisiae</i> and <i>Synechococcus
| + | |
| − | elongatus</i> sp. PCC 7942 based on the availability of essential
| + | |
| − | nutrients such as nitrogen(7), phosphate(8) and carbon(9).
| + | |
| − | The aim of system 2 is the dependency of <i>E.coli</i>, <i>S. cerevisiae</i>
| + | |
| − | and <i>S. elongatus</i>, achieved through the exploitation of
| + | |
| − | auxotrophies. <i>S. cerevisiae</i> is in our case auxotrophic for lysine
| + | |
| − | which is produced by <i>E.coli</i>(10). At the same time, <i>E.coli</i> has
| + | |
| − | an auxotrophy for leucine, which, in turn, is provided by
| + | |
| − | <i>S. cerevisiae</i>. <i>S. elongatus</i> shows no auxotrophy and enriches the
| + | |
| − | media with the monosaccharides glucose and fructose. This way
| + | |
| − | <i>E.coli</i> and <i>S. cerevisiae</i> are regulating each other and do not
| + | |
| − | overgrow S. elongatus. Each organism is responsible for the
| + | |
| − | production of one substance, amino acid or glucose, and thus
| + | |
| − | makes the other organism dependent on it.
| + | |
| − | System 3 is utilising the quorum sensing mechanism to regulate
| + | |
| − | cell density in the population by using bacterial communication
| + | |
| − | molecules. <i>E.coli</i> regulates its own cell density by expressing
| + | |
| − | a lysis gene after induction by AHL1 quorum sensing
| + | |
| − | molecules(11). For <i>S. cerevisiae</i> two different systems were
| + | |
| − | introduced. The first one utilises the fact that upon recognition
| + | |
| − | of the complementary mating type factor pheromone (MAT `a` for
| + | |
| − | `alpha` yeast) yeast cells go into a cell cycle arrest and stop
| + | |
| − | growing(12). The second system depends on the design of a
| + | |
| − | synthetic promoter that activates another lysis gene after the
| + | |
| − | recognition of AHL2 molecules(13).
| + | |
| − | </p>
| + | |
| − | <p>The following box provides a graphical overview as well as a summery
| + | |
| − | of the role of each organism in the system for each Level:
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <section class ="levelbox">
| + | |
| − |
| + | |
| − | <!-- Level 1 -->
| + | |
| − | <div align="center">
| + | |
| − | <div id="Level1" class="tabcontent">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/a/a2/T--Duesseldorf--1s.png", width=100%>
| + | |
| − | </div>
| + | |
| | | | |
| − | <div id="E.Coli" class="tabcontent">
| + | <h1>PROJECT DESCRIPTION</h1> |
| − | <h3><i>E.coli</i>:</h3>
| + | |
| − | <p>A synthetic cluster of 6 genes from different organisms
| + | |
| − | (<i>Acidovorax avenae, E. coli, Pseudomonas sp., Rhodococcus sp.,
| + | |
| − | S. cerevisiae</i>) used for the cleavage of melamin to ammonia and
| + | |
| − | carbon dioxide. The ammonia diffuses out of the cell.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| | | | |
| − | <div id="S.cerevisiae" class="tabcontent">
| + | <h2>About</h2> |
| − | <h3><i>S. cerevisiae</i>:</h3>
| + | <p class=details> Scientists all over the world in different fields are using microorganisms for their research - that is nothing new. But most of them are using monocultures and therefore miss thousands of possibilities which arise in co-cultures. It is estimated that less than 1% of all microorganisms have been successfully cultivated<sup>1</sup>. A reason for that might be the very artificial conditions in the laboratory. In nature no organism is completely isolated, but rather lives in very complex systems. Scientist have only just begun to investigate the complex interaction between different microorganisms but yet often fail at the cultivation. Easily obtainable and stable co-cultures would allow research of yet uninvestigated species and might give further insight into cell-cell-interactions between microorganisms<sup>2</sup>. One problem of isolated cultures is the regular use of antibiotics as selective pressure and prevention of contaminations which leads towards multiple resistances. Here as well a co-culture with nutrient dependencies instead of higher antibiotic concentration is a better alternative. |
| − | <p>With the gene ptxD from <i>P. putida S. cerevisiae</i> converts the
| + | Pharmacotherapeutic companies spend a lot of money on developing new antibiotics, while in co-culturing conditions some microorganisms show differential gene expression and might produce new antibiotics on their own<sup>3</sup>. In general, co-cultures might provide a great opportunity as a new method for cultivation and the production of new antibiotics or industrially interesting products. There is yet a lot of potential for new co-cultures and we - Team iGEM 2018 HHU - want to tackle that challenge. |
| − | unusable phosphite into phosphate. The phosphate exporter XPR1
| + | </p> |
| − | from H. sapiens may help to increase the extracellular phosphate
| + | <br> |
| − | concentration.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| | | | |
| − | <div id="S.elongatus" class="tabcontent">
| + | <h2>The Challenges</h2> |
| − | <h3><i>S. elongatus</i>:</h3>
| + | <p class=details> Before starting research, first the safety must be ensured. But this is a self solving problem, since the organisms are only able to live in a community that is designed by the researcher itself. That includes a detailed control of the microorganisms, the cell density, their behavior and environmental conditions. For industrial usage the production must be scalable and high throughput must be guaranteed as well. Furthermore, general technologies for a normed use for research projects that are also easily applicable for large scale production must be achieved< sup> 4</ sup> . One goal would be the reduction and simplification of processing steps and easy-to-use protocols for stable co-cultures to make cultivation and production faster and cheaper. Other requirements for making co-cultures a profitable alternative to isolated culturing are big product diversity and efficient extraction of them. For scientists the possibility to build complex systems and methods for big data collection are desirable and highly demanded. |
| − | <p> Converting sucrose, which is naturally produced by photosynthesis, | + | </p> |
| − | into fructose and glucose by an invertase encoded by the gene
| + | <br> |
| − | invA. Glucose will be secreted into the media with the help of an
| + | |
| − | exporter, encoded by glf, and thus providing enough glucose for
| + | |
| − | the whole trinity. The genes are taken from the <i>Z. mobilis</i> genome.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| | | | |
| − | <button class="tablink" onclick="openCity('Level1', this, 'gray')" id="defaultOpen">Level 1:</button>
| + | <h2>The Project</h2> |
| − | <button class="tablink" onclick="openCity('E.Coli', this, 'gray')">E.coli:</button>
| + | <p class=details> Since our co-cultures should be universally usable for laboratories all over the world, we plan to design it as easy and modular as possible. Therefore, the Golden Gate modular cloning standard techniques (MoClo) were used besides Gibson Assembly for our cloning steps, which allows exchange of all parts to adjust the co-culture to every possible condition<sup>5,6</sup>. |
| − | <button class="tablink" onclick="openCity('S.cerevisiae', this, 'gray')">S. cerevisiae:</button>
| + | |
| − | <button class="tablink" onclick="openCity('S.elongatus', this, 'gray')">S. elongatus:</button>
| + | |
| − | </div>
| + | |
| | | | |
| − | <!-- Level 2 -->
| + | We envision a standardised system, a three-way co-culture with a selection of organisms and regulations. `Trinity` is going to incorporate different dependencies between the organisms in order to control their growth and enable them to adjust each others behaviour. |
| − | <div align="center">
| + | Project Trinity is divided into three systems. |
| − | <div id="Level2" class="tabcontent">
| + | Our system 1 deals with the dependence of the organisms <i>Escherichia coli</i>, <i>Saccharomyces cerevisiae</i> and <i>Synechococcus elongatus</i> sp. PCC 7942 based on the availability of essential nutrients such as nitrogen<sup>7</sup>, phosphate<sup>8</sup> and carbon<sup>9</sup>. The aim of system 2 is the dependency of <i>E.coli</i>, <i>S. cerevisiae</i> and <i>S. elongatus</i> , achieved through the exploitation of auxotrophies. <i>S. cerevisiae</i> is in our case auxotrophic for lysine which is produced by E.coli<sup>10</sup>(. At the same time, E.coli has an auxotrophy for leucine, which, in turn, is provided by <i>S. cerevisiae</i>. <i>S. elongatus</i> shows no auxotrophy and enriches the media with the monosaccharides glucose and fructose. This way <i>E.coli</i> and <i>S. cerevisiae</i> are regulating each other and do not overgrow <i>S. elongatus</i>. Each organism is responsible for the production of one substance, amino acid or glucose, and thus makes the other organism dependent on it. System 3 is utilising the quorum sensing mechanism to regulate cell density in the population by using bacterial communication molecules. <i>E.coli</i> regulates its own cell density by expressing a lysis gene after induction by AHL1 quorum sensing molecules<sup>11</sup>. For <i>S. cerevisiae</i> two different systems were introduced. The first one utilises the fact that upon recognition of the complementary mating type factor pheromone (MAT `a` for `alpha` yeast) yeast cells go into a cell cycle arrest and stop growing<sup>12</sup>. The second system depends on the design of a synthetic promoter that activates another lysis gene after the recognition of AHL2 molecules<sup>13</sup>. |
| − | <img src="https://static.igem.org/mediawiki/2018/1/13/T--Duesseldorf--2s.png", style=" width:100%">
| + | |
| − | </div>
| + | |
| | | | |
| − | <div id="E.Coli2" class="tabcontent">
| + | The following box provides a graphical overview as well as a summery of the role of each organism in the system for each Level: . |
| − | <h3><i>E.coli</i>:</h3>
| + | </p> |
| − | <p>Leucine auxotrophic <i>E.coli</i> are engineered to produce lysine for
| + | |
| − | <i>S. cerevisiae</i>. For optimization of leucine production a not-feedback
| + | |
| − | inhibiting gene lysC from C. glutamicum and the gene ddh are
| + | |
| − | introduced to attain the highest possible leucine yield. That allows
| + | |
| − | the auxotrophic character as a dependency as well as a selection
| + | |
| − | marker.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| | | | |
| − | <div id="S.cerevisiae2" class="tabcontent">
| + | <br></br> |
| − | <h3><i>S. cerevisiae</i>:</h3>
| + | <h2>Our Trinity</h2> |
| − | <p> Is auxotroph for lysine, thus dependent on the production of lysine by | + | <p class=details> Since our co-cultures should be universally usable for laboratories all over the world, we plan to design it as easy and modular as possible. Therefore, the Golden Gate modular cloning standard techniques (MoClo) were used besides Gibson Assembly for our cloning steps, which allows exchange of all parts to adjust the co-culture to every possible condition< sup> 5,6</ sup>. |
| − | <i>E.coli</i>, but on the other hand produces leucine for <i>E.coli</i>. Therefore
| + | |
| − | Leu2 is overexpressed to reach a sufficient concentration of leucine
| + | |
| − | for <i>E.coli</i> to grow.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| | | | |
| − | <div id="S.elongatus2" class="tabcontent">
| + | We envision a standardised system, a three-way co-culture with a selection of organisms and regulations. `Trinity` is going to incorporate different dependencies between the organisms in order to control their growth and enable them to adjust each others behaviour. |
| − | <h3><i>S. elongatus</i>:</h3>
| + | Project Trinity is divided into three systems. |
| − | <p>As in level 1 <i>S. elongatus</i> is responsible for the carbon source by
| + | Our system 1 deals with the dependence of the organisms <i>Escherichia coli</i>, <i>Saccharomyces cerevisiae</i> and <i>Synechococcus elongatus</i> sp. PCC 7942 based on the availability of essential nutrients such as nitrogen<sup>7</sup>, phosphate<sup>8</sup> and carbon<sup>9</sup>. The aim of system 2 is the dependency of <i>E.coli</i>, <i>S. cerevisiae</i> and <i>S. elongatus</i> , achieved through the exploitation of auxotrophies. <i>S. cerevisiae</i> is in our case auxotrophic for lysine which is produced by E.coli<sup>10</sup>. At the same time, <i>E.coli</i> has an auxotrophy for leucine, which, in turn, is provided by <i>S. cerevisiae</i>. <i>S. elongatus</i> shows no auxotrophy and enriches the media with the monosaccharides glucose and fructose. This way <i>E.coli</i> and <i>S. cerevisiae</i> are regulating each other and do not overgrow <i>S. elongatus</i>. Each organism is responsible for the production of one substance, amino acid or glucose, and thus makes the other organism dependent on it. System 3 is utilising the quorum sensing mechanism to regulate cell density in the population by using bacterial communication molecules. <i>E.coli</i> regulates its own cell density by expressing a lysis gene after induction by AHL1 quorum sensing molecules<sup>11</sup>. For <i>S. cerevisiae</i> two different systems were introduced. The first one utilises the fact that upon recognition of the complementary mating type factor pheromone (MAT `a` for `alpha` yeast) yeast cells go into a cell cycle arrest and stop growing<sup>12</sup>. The second system depends on the design of a synthetic promoter that activates another lysis gene after the recognition of AHL2 molecules<sup>13</sup>. |
| − | producing sugar using sunlight and carbon dioxide.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| | | | |
| − | <button class="tablink" onclick="openCity('Level2', this, 'gray')" id="defaultOpen">Level 2:</button>
| |
| − | <button class="tablink" onclick="openCity('E.Coli2', this, 'gray')">E.coli:</button>
| |
| − | <button class="tablink" onclick="openCity('S.cerevisiae2', this, 'gray')">S. cerevisiae:</button>
| |
| − | <button class="tablink" onclick="openCity('S.elongatus2', this, 'gray')">S. elongatus:</button>
| |
| − | </div>
| |
| − |
| |
| − | <!-- Level 3 -->
| |
| − | <div align="center">
| |
| − | <div id="Level1" class="tabcontent">
| |
| − | <img alt="Level 3" style = "width:q00%;">
| |
| − | </div>
| |
| | | | |
| − | <div id="E.coli3" class="tabcontent">
| + | <br><br> |
| − | <h3><i>E.coli</i>:</h3>
| + | |
| − | <p>For cell-density control, <I>E.coli</i> harbours the gene luxI producing AHL
| + | |
| − | constantly and the gene luxR which continuously expresses the LuxR
| + | |
| − | protein. This protein recognizes the AHL molecule and then activates
| + | |
| − | the Lux promoter, which expresses the lysis gene<I> phiX174E</I>. Thus the
| + | |
| − | population reduces itself after a threshold is reached.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| | | | |
| − | <div id="S.cerevisiae3" class="tabcontent">
| + | <h3>References</h3> |
| − | <h3><i>S. cerevisiae</i>:</h3>
| + | <ol type="1"> |
| − | <p>Using the natural mating type system, a MAT “𝜶” strain goes into
| + | |
| − | cell cycle arrest when recognising an “a” pheromone. This way, an “a”
| + | |
| − | producing alpha strain regulates itself at a high cell density.
| + | |
| − | A different approach is a synthetic promoter, which makes the yeast
| + | |
| − | compatible to the bacterial quorum sensing system. The synthetic
| + | |
| − | promoter is activated by luxR after binding the AHL2 molecule and
| + | |
| − | then activates the lysis gene.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div id="S.elongatus3" class="tabcontent">
| + | |
| − | <h3><i>S. elongatus</i>:</h3>
| + | |
| − | <p>Since <i>S. elongatus</i> has the longest reproduction time of the three
| + | |
| − | organisms, no quorum sensing specific changes are required,
| + | |
| − | concerning the growth rate of this organism. However, the strain,
| + | |
| − | which supplies glucose to the media is used to supply the other
| + | |
| − | organisms with the required carbon source.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <button class="tablink" onclick="openCity('Level3', this, 'gray')" id="defaultOpen">Level 3:</button>
| + | |
| − | <button class="tablink" onclick="openCity('E.coli3', this, 'gray')">E.coli:</button>
| + | |
| − | <button class="tablink" onclick="openCity('S.cerevisiae3', this, 'gray')">S. cerevisiae:</button>
| + | |
| − | <button class="tablink" onclick="openCity('S.elongatus3', this, 'gray')">S. elongatus:</button>
| + | |
| − | </div>
| + | |
| − | </section>
| + | |
| − | | + | |
| − |
| + | |
| − | <section class="references">
| + | |
| − | <h4>References: <br></h4>
| + | |
| − | | + | |
| | <li>Katz, Micah, Bradley M. Hover, and Sean F. Brady. "Culture-independent | | <li>Katz, Micah, Bradley M. Hover, and Sean F. Brady. "Culture-independent |
| | discovery of natural products from soil metagenomes." Journal of industrial | | discovery of natural products from soil metagenomes." Journal of industrial |
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| | </li> | | </li> |
| | </ol> | | </ol> |
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