Difference between revisions of "Team:Goettingen/Results"

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<h4> Project Achievements </h4>
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<a href="" onClick=" $('#menu4').slideToggle(300, function callback() {  }); return false;"><h1>BioBricks</h1></a>
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<h5>What should this page contain?</h5>
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<div id="menu4">
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<p>Here you can describe the results of your project and your future plans. </p>
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<ul>
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<li> Clearly and objectively describe the results of your work.</li>
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<li> Future plans for the project </li>
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<li> Considerations for replicating the experiments </li>
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</ul>
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
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<p>
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    <strong>Dockerin Biobricks</strong>
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</p>
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<p>
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The dockerins ACEL (<em>Acetivibrio cellulolyticus</em>), BCEL (<em>Bacteroides cellulosolvens</em>), CCEL (<em>Clostridium cellulolyticum</em>) and CTHE (<em>Clostridium thermocellum</em>) were amplified from pBAD_ACEL, pBAD_BCEL, pBAD_CCEL and pBAD_CTHE with primers to add the desired <em>Eco</em>RI and   <em>Pst</em>I restriction sites by PCR. Primers contained restriction sites for <em>Eco</em>RI and <em>Pst</em>I in order to make them compatible for
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    insertion into the iGEM shipping vector pSB1C3.
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</p>
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<p>
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    After purification, the PCR products were restricted with <em>Eco</em>RI and <em>Pst</em>I, as well as pSB1C3. Afterwards the restricted dockerins were
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    ligated into pSB1C3 by T4 ligation (sticky end ligation) and transformed into <em>E. coli</em> TOP10.
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</p>
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<p>
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    Ligation into pSB1C3 was followed according to the protocol in the methods collection. After over-night incubation colonies were picked, plasmids extracted
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    with the QIAGEN QIAprep Spin Miniprep Kit and test restricted with <em>Eco</em>RI (pSB1C3_CCEL and pSB1C3_ACEL) and <em>Eco</em>RI and <em>Pst</em>I
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    (pSB1C3_BCEL) (Fig.1). We lost CTHE due to failing transformations at this point.
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</p>
  
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<ul>
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</html>
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<li>A list of linked bullet points of the successful results during your project</li>
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[[File:Rest_control_pSB1C3_ACEL_CCEL_BCEL.jpg|600px|thumb|center|]]
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
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<html>
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</ul>
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<p>
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    Once restriction controls showed the correct bands, pSB1C3_CCEL, pSB1C3_ACEL and pSB1C3_BCEL were sent for sequencing.
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</p>
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<p>
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    <strong>Colour BioBricks</strong>
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</p>
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<p>
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    We also wanted to improve already existing BioBricks by fusing our dockerins to the colours eforRed (BBa_K592012) and amilCP (BBa_K592009) that were
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    submitted from the University of Uppsala (Sweden) in 2011.
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</p>
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<p>
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    So we chose to fuse eforRed to BCEL and amilCP to CCEL. Since the plasmids of the two colour proteins were not distributed with the current plate of the
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    iGEM Distribution Kit, we ordered those parts including the right restriction sites as gBlocks from IDT.
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</p>
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<p>
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    As a first step eforRed and amilCP were restricted with <em>Kpn</em>I and <em>Sac</em>I to make them compatible with our dockerins in pBAD.
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</p>
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<p>
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    After purification restricted colours were ligated into pBAD_BCEL and pBAD_CCEL by T4 ligation (sticky end ligation) and transformed into <em>E. coli</em>
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    TOP10.
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</p>
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<p>
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    Ligation into pBAD was followed according to the protocol in the methods collection. After over-night incubation colonies were picked, plasmids extracted
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    with the QIAGEN QIAprep Spin Miniprep Kit and test restricted with <em>Kpn</em>I (Fig.2).
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</p>
  
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</html>
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[[File:TeamGoettingen2015_Rest_control_pBAD_eforRed_BCEL_and_amilCP_CCEL_Goettingen2015.jpeg|600px|thumb|center|]]
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<html>
  
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<h4>Inspiration</h4>
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<p>
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<p>See how other teams presented their results.</p>
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    Once restriction controls showed the correct bands, pBAD_amilCP_CCEL #1 and pBAD_eforRed_BCEL were sent for sequencing. Sequencing showed that both
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<ul>
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    plasmids were correct.
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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</p>
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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
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<p>
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
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    To make our constructs compatible with the iGEM shipping vector pSB1C3 the desired <em>Eco</em>RI and <em>Pst</em>I restrictions sites were added by PCR.
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</ul>
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</p>
 +
<p>
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    After purification, the PCR products were restricted with <em>Eco</em>RI and <em>Pst</em>I, the same with pSB1C3, and afterwards ligated into pSB1C3 by T4
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    ligation (sticky end ligation) and transformed into <em>E. coli</em> TOP10.
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</p>
 +
<p>
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    After over-night incubation colonies were picked, plasmids extracted with the QIAGEN QIAprep Spin Miniprep Kit and test restricted with <em>Eco</em>RI
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    (Fig.3). Due to the reason that our dockerin CCEL contains an internal <em>Pst</em>I restriction site we continued working only with the eforRed_BCEL
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    construct.
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</p>
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</html>
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[[File:TeamGoettingen2015_Rest_control_pSB1C3_eforRed_BCEL_Goettingen2015.jpeg|600px|thumb|center|]]
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<html>
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<p>
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    Once restriction controls showed the correct band, pSB1C3_eforRed_BCEL was sent for sequencing. Sequencing showed that the plasmids were correct.
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</p>
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<p>
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    <strong>RESULTS</strong>
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</p>
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<p>
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    Sequencing showed that the dockerins ACEL and CCEL contain an internal <em>Pst</em>I restriction site. Therefore all the constructs containing ACEL or CCEL
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    showed in the end truncated dockerin sequences and could not be send in as BioBricks.
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</p>
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<p>
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    We also build a construct where the enzyme esterase was fused to our CCEL dockerin (pBAD_Est_CCEL) following the same strategy. But again, due to the
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    reason that our dockerin CCEL contains an internal <em>Pst</em>I restriction site we had to stop our work at this point.
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</p>
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<p>
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    Furthermore we collaborated with the current iGEM team of Aachen and tried to fuse three of their enzymes to our constructs (see Collaboration link) but
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    could not finish our work here.
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</p>
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<p>
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    <strong>ACHIEVENTS<a name="_GoBack"></a></strong>
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</p>
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<p>
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    <strong>Sequences of pSB1C3_BCEL and pSB1C3_eforRed_BCEL were correct and submitted as BioBricks to iGEM:</strong>
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</p>
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<p>
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    pSB1C3_BCEL (BBa_1865000)
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</p>
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<p>
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    pSB1C3_eforRed_BCEL (BBa_1865001)
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</p>
  
 
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Revision as of 18:06, 18 September 2015



Project Results

Transformation Efficiency Kit, RFP construct (iGEM)

RFP

Esterase and Phosphatase

BioBricks