Team:Cambridge-JIC

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                        <h1>Cambridge JIC</h1>
 
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                        <h4>Welcome to the page for the Cambridge University team,<br />affiliated with the John Innes Centre in Norwich.</h4>
 
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                    <h2 class="section-heading">mösbi - a plant biosensor for everyone</h2>
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                    <p class="lead"><b>mӧsbi</b> stands for <b>m</b>archantia <b>o</b>pen-<b>s</b>ource <b>bi</b>osensor. mӧsbi encapsulates the team's vision of creating a user-friendly, open-source biosensor using the liverwort <i>Marchantia polymorpha</i>. With its sturdy genetic framework and modularity, mӧsbi represents a new step in popularising synthetic biology and making it accessible to a large audience. Click <a href="https://2014.igem.org/Team:Cambridge-JIC/Project/Overview">here</a> to find out more.</p>
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                    <h2 class="section-heading"><i>Marchantia polymorpha</i> as a new chassis</h2>
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                    <p class="lead"> <i><b>Marchantia polymorpha</b></i> is our novel, eukaryotic multicellular chassis. Being a liverwort, it is one of the most primitive land plants around. Its small size and relative genetic simplicity make it easy to work with and an exciting new model organism in synthetic biology. Content to grow on agar plates, marchantia can be bioengineered in a standard lab with minimal extra equipment. Click <a href="https://2014.igem.org/Team:Cambridge-JIC/Marchantia/Background">here</a> to find out more about the plant and our work to develop the chassis; and <a href="https://2014.igem.org/Team:Cambridge-JIC/Guide">here</a> to learn how to get started using <i>Marchantia</i> in your own iGEM project.</p>
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<h1> Chromoprotein Constructs</h1>
 
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<h2> Design </h2>
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<h4> Aim of the construct </h4>
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Chromoproteins represent ideal reporter genes. Unlike fluorescent protein, from which chromoproteins were originally derived, they require no specialised optical equipment for detection. Our aim is to use a selection of such proteins, donated by the iGEM 2012 Uppsala team, as our most basic and main output in regards to the Marchantia framework.
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We decided to test the following chromoprotein constructs:
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<li> 35s - eforRed - nosT</li>
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<li> 35s - eforRed - N7 - nosT</li>
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                    <h2 class="section-heading">Results</h2>
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<li> 35s - amilCP - N7 - nosT</li>
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                    <p class="lead">We successfully transformed hundreds of individual plants, resulting in the first expression of chromoproteins in <i>Marchantia</i>, and probably the first expression of chromoproteins in any plant. We successfully built an arduino-controlled growth chamber, characterised an enzyme new to iGEM and amended the community's knowledge of an existing registry entry. A known <i>Marchantia</i> promoter was also added to the registry, along with 27 candidate promoters from the <i>Marchantia</i>genome. They are part of a <i>Marchantia</i> starter kit part collection that also includes terminators, direction sequences, etc. Finally, we submitted an RFC with collaboration of two other iGEM teams, establishing a unified Type IIS based grammar for plant synthetic biologist: PlantSyntax. Click <a href="https://2014.igem.org/Team:Cambridge-JIC/Results/Lab">here</a> for more result details, and <a href="https://2014.igem.org/Team:Cambridge-JIC/ExecSummary">here</a> for our executive summary.</p>
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<li> 35s - tsPurple - nosT</li>
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<li> 35s - tsPurple - N7 - nosT</li>
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<br></br>By expressing the chromoproteins in the pGreen vector with the 35s promoter and the nosT terminaor, we control for the possiblity of the promoter/terminator not functioning in our chassis.
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The N7 fragment allows for the selected constructs to localise chromoprotein to the nucleus. It is believed by Bernardo Pollak that this may end up increasing the likelihood of detecting the chromoproteins with the naked eye.
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                    <h2 class="section-heading">Our team</h2>
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                    <p class="lead">We are 9 Cambridge science undergraduates from various backgrounds and with all kinds of fascinating and curious interests. Click <a href="https://2014.igem.org/Team:Cambridge-JIC/Team">here</a> to learn more about the team.</p>
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<div id="The plasmid" align="left">
 
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<h4> End goal plasmid </h4>
 
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<li> insert image here </li>
 
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<h2> Experimentation (up to date)</h2>
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<h4>PCR</h4>
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Attempt 1:
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<p>Date: 18.07.2014</p>
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<p>UIDs of primers/plasmids used:</p>
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<ul><li>Backbone Fragment 1 (35s - Hyg promoter): B14, P16, P14. Tubes: CpI 1</li>
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<li>Backbone Fragment 2(Hyg promoter - nosT): B14, P15, P17. Tubes: CpI 2</li>
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<li>N7 fragment: B15, P13, P22. Tubes: CpI 3</li>
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<li>eforRed gene(cytoplasmic): B13, P1, P2. Tubes: CpI 4</li>
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<li> eforRed gene(nuclear): B13, P1, P3. Tubes: CpI 5</li>
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<li> amilCP gene(nuclear): B10, P4, P5. Tubes: CpI 6</li>
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<li> tsPurple gene(cytoplasmic): B12, P6, P7. Tubes: CpI 7</li>
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<li> tsPurple gene(nuclear): B12, P6, P8. Tubes: CpI 8</li>
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<li> asPink gene(nuclear): B9, P9, P10. CpI 9</li>
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<li> aeBlue gene(nuclear): B11, P11, P12. Tubes: CpI 10</li></ul>
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<p>After running the gel, the concentrations of CpI 1, CpI 2 and CpI 3 were much lower compared to the chromoprotein genes (see gel picture). Amplification of CpI 3 failed. The problem was an alteration to the primers necessary for amplification. Re-ran PCR for CpI 3. Modification: Appropriate primers (P13 & P22) and the extension phase of PCR has been reduced from 2 min to 15 sec since N7 is only ~300bp long.
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The DNA was then purified using the QIAGEN QiaQuick Gel Purification Kit. The DNA was eluted in 50 ul of EB buffer. Due to the large elution volume and the lower yield of the backbone DNA, the concentrations (measured using Nanodrop) were lower than the suggested 10ng/ul threshold for Gibson.
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<p><a href="http://pcrspreadsheet.com">PCR Excell Protocol</a></p>
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<p><a href="http://google.com/GEL_27_07_2014">Gel Picture</a></p>
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Attempt 2:
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<p>Date: 21.07.2014</p>
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<p>UIDs of primers/plasmids used:</p>
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<ul><li>Backbone Fragment 1 (35s - Hyg promoter): B14, P16, P14. Tubes: CpII 1</li>
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<li>Backbone Fragment 2(Hyg promoter - nosT): B14, P15, P17. Tubes: CpII 2</li>
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<li>N7 fragment: B15, P13, P22. Tubes: CpII 3</li>
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<li>eforRed gene(cytoplasmic): B13, P1, P2. Tubes: CpII 4</li>
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<li> eforRed gene(nuclear): B13, P1, P3. Tubes: CpII 5</li>
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<li> amilCP gene(nuclear): B10, P4, P5. Tubes: CpII 6</li>
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<li> tsPurple gene(cytoplasmic): B12, P6, P7. Tubes: CpII 7</li>
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<li> tsPurple gene(nuclear): B12, P6, P8. Tubes: CpII 8</li>
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<li> asPink gene(nuclear): B9, P9, P10. CpII 9</li>
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<li> aeBlue gene(nuclear): B11, P11, P12. Tubes: CpII 10</li></ul>
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<p>Due to the low yield of the DNA purification fro Attempt 1, the entire PCR was repeated. Upon gel separation we realised the PCRs were nearly identical (see Gel Picture), which is what we were expecting. However, the DNA was euted into 10ul of EB buffer instead of 50ul during the gel purification step, which increased the concentrations of CpII 4-10 significantly. However, CpII 1-3 concentrations were still below the 10 ng DNA/ul range. </p></ul>
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<p><a href="http://pcrspreadsheet.com">PCR Excell Protocol</a></p>
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<p><a href="http://google.com/GEL_27_07_2014">Gel Picture</a></p>
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Attempt 3:
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<p>Date: 22.07.2014</p>
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<p>UIDs of primers/plasmids used:</p>
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<ul><li>Backbone Fragment 1 (35s - Hyg promoter): B14, P16, P14. Tubes: CpIII 1</li>
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<li>Backbone Fragment 2 - Full Size(Hyg promoter - nosT): B14, P15, P17. Tubes: CpIII 2 RBLB</li>
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<li>Backbone Fragment 2 - LB(Hyg promoter - KanR): B14, P18, P17. Tubes: CpIII 2 LB</li>
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<li>Backbone Fragment 2 - RB(KanR - nosT): B14, P15, P19. Tubes: CpIII 2 RB</li>
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<li>N7 fragment: B15, P13, P22. Tubes: CpIII 3</li>
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<p>The backbone fragments were re-aplified to allow for future assemblies to be made. The PCR was altered to run for 35 amplification cycles instead of 30, which increase the yield of DNA. </p></ul>
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<p><a href="http://pcrspreadsheet.com">PCR Excell Protocol</a></p>
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<p><a href="http://google.com/GEL_27_07_2014">Gel Picture</a></p>
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<h4> Gibson </h4>
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Attempt 1:
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<li>Date: 21.07.2014</li>
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<li>End date/time:</li>
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                    <p class="lead">We are a multidisciplinary group of 9 enthusiastic Cambridge science undergraduates. Click <a href="https://2014.igem.org/Team:Cambridge-JIC/Team">here</a> to meet the team.</p>
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Latest revision as of 01:17, 18 October 2014

Cambridge iGEM 2014

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mösbi - a plant biosensor for everyone

mӧsbi stands for marchantia open-source biosensor. mӧsbi encapsulates the team's vision of creating a user-friendly, open-source biosensor using the liverwort Marchantia polymorpha. With its sturdy genetic framework and modularity, mӧsbi represents a new step in popularising synthetic biology and making it accessible to a large audience. Click here to find out more.

Marchantia polymorpha as a new chassis

Marchantia polymorpha is our novel, eukaryotic multicellular chassis. Being a liverwort, it is one of the most primitive land plants around. Its small size and relative genetic simplicity make it easy to work with and an exciting new model organism in synthetic biology. Content to grow on agar plates, marchantia can be bioengineered in a standard lab with minimal extra equipment. Click here to find out more about the plant and our work to develop the chassis; and here to learn how to get started using Marchantia in your own iGEM project.

Results

We successfully transformed hundreds of individual plants, resulting in the first expression of chromoproteins in Marchantia, and probably the first expression of chromoproteins in any plant. We successfully built an arduino-controlled growth chamber, characterised an enzyme new to iGEM and amended the community's knowledge of an existing registry entry. A known Marchantia promoter was also added to the registry, along with 27 candidate promoters from the Marchantiagenome. They are part of a Marchantia starter kit part collection that also includes terminators, direction sequences, etc. Finally, we submitted an RFC with collaboration of two other iGEM teams, establishing a unified Type IIS based grammar for plant synthetic biologist: PlantSyntax. Click here for more result details, and here for our executive summary.

Our team

We are 9 Cambridge science undergraduates from various backgrounds and with all kinds of fascinating and curious interests. Click here to learn more about the team.

Retrieved from "http://2014.igem.org/Team:Cambridge-JIC"