Team:BostonU/Results

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JUMP TO...<br>
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<a href="#bu-wellesley_wiki_content">Top</a><br>
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<a href="#DVResults">Destination Vectors</a><br>
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<a href="#BBtoMC">BioBricks to MoClo</a><br>
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<a href="#Level0Parts">Creating Level 0 Parts</a><br>
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<a href="#Level1Parts">Creating Level 1 Parts</a><br>
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<h4>Results Summary</h4>
<h4>Results Summary</h4>
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<h9>Creating Destination Vectors<h9><ul>
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<div id="DVResults">
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<h9>Creating Destination Vectors<h9>
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<h7><br>Below are the step by step results we obtained in making all of our destination vectors:<br>
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<h7><p dir="ltr">Below are the step by step results we obtained in making all of our destination vectors:<br>
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<li>We first transformed the destination vectors into cells on to plates with IPTG/X-Gal for blue white screening. In this case, we wanted to see blue cells, indicative of the presence of LacZ. Our destination vectors also varied in whether the backbone is phosphorylated or dephosphorylated. In this picture, the dephosphorylated showed higher  transformation efficiency by preventing recircularization of the backbone, yielding more cells transformed with the LacZ as part of the destination  vector.
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First, we transformed the destination vectors into cells onto plates with IPTG/X-Gal for blue white screening. In this case, we wanted to see blue cells, indicative of the presence of LacZ. Our destination vectors also varied in whether the backbone is phosphorylated or dephosphorylated. In this picture, the dephosphorylated showed higher  transformation efficiency by preventing recircularization of the backbone, yielding more cells transformed with the LacZ as part of the destination  vector.
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<img src="https://static.igem.org/mediawiki/2012/a/ac/Sum2.png" width="500px">
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<li>We checked whether the destination vectors contained the LacZ fragment amplified with the moclo fusion sites by running digest of cut vs uncut. In the gel picture, each SM refers to a different destination vector and the C and UC means cut and uncut, respectively.  
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We checked whether the destination vectors contained the LacZ fragment amplified with the moclo fusion sites by running digest of cut vs uncut. In the gel picture, each SM refers to a different destination vector and the C and UC means cut and uncut, respectively.  
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<p><center>
<img src="https://static.igem.org/mediawiki/2012/e/e3/Sum1.png" width="500px">
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<li>The final step is sequencing the destination vectors to confirm the correct orientation of LacZ. In the nucleotide sequence file and the complementary diagram, the LacZ sequence, 2 fusion sites and restriction sites are highlighted in different colors.  
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The final step is sequencing the destination vectors to confirm the correct orientation of LacZ. In the nucleotide sequence file and the complementary diagram, the LacZ sequence, 2 fusion sites and restriction sites are highlighted in different colors.  
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<Li>The trace file shows confidence of the sequencing.
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The trace file shows confidence of the sequencing.
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<img src="https://static.igem.org/mediawiki/2012/0/04/Sum4.png" width="350px"><br>
 
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<div id="BBtoMC">
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<h9>Converting Biobrick to Moclo Parts:<h9>
<h9>Converting Biobrick to Moclo Parts:<h9>
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<h7><p dir="ltr">We first added fusion sites to Biobrick parts through PCR. The primers contained the sequence for the fusion sites as part its overhang. In the process we used regular PCR amplification and also ligation PCR.
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The first picture refers to promoters R0010 and R0079 with the added fusion sites
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<li>We first added fusion sites to Biobrick parts through PCR. The primers contained the sequence for the fusion sites as part its overhang. In the process we used regular PCR amplification and also ligation PCR.
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<li>The first picture refers to promoters R0010 and R0079 with the added fusion sites
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<img src="https://static.igem.org/mediawiki/2012/5/58/Sum5.png" width="300px"><br>
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<img src="https://static.igem.org/mediawiki/2012/5/58/Sum5.png" width="300px">
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<li>The second picture is the result of ligation PCR of the J series promoters.
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The second picture is the result of ligation PCR of the J series promoters.
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<div id="Level0Parts">
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<h9>Creating Moclo Level 0 Parts</h9>
<h9>Creating Moclo Level 0 Parts</h9>
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<h7><p dir="ltr">Afterwards we transformed and plated cells with level 0 Moclo parts on to IPTG/X-Gal plates for blue white screening where we look for white colonies. In the picture we see white colonies, indicating the LacZ was cut out of the destination vector and replaced by parts with the Moclo fusion sites.
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<li>Afterwards we transformed and plated cells with level 0 Moclo parts on to IPTG/X-Gal plates for blue white screening where we look for white colonies. In the picture we see white colonies, indicating the LacZ was cut out of the destination vector and replaced by parts with the Moclo fusion sites.
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<img src="https://static.igem.org/mediawiki/2012/6/6a/Sum7.png" width="600px">
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We confirmed the Moclo level 0 parts through sequencing. Below there is an example of the sequence file we received for a promoter, RBS, gene and terminator. The files were edited with highlights to indicate the correct locations of the part, fusion sites and the restriction sites.
We confirmed the Moclo level 0 parts through sequencing. Below there is an example of the sequence file we received for a promoter, RBS, gene and terminator. The files were edited with highlights to indicate the correct locations of the part, fusion sites and the restriction sites.
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<img src="https://static.igem.org/mediawiki/2012/7/74/Rbs.png" width="600px">
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<img src="https://static.igem.org/mediawiki/2012/f/fd/TERMINATOR.png" width="600px">  
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<h9>Creating Level 1 Moclo Parts<h9>
<h9>Creating Level 1 Moclo Parts<h9>
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<li>We combined level 0 promoter, RBS, gene and terminator in a one pot reaction. Which then we transformed, then screened for white colonies on IPTG/X-Gal plates.
 
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<li>We checked the level 1s through a double digest and checking it on a gel. The gel below shows bands at 2 kb to be the destination vector and the smaller bands at 75~800 bp indicating the presence of the transcriptional unit.
 
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<img src="https://static.igem.org/mediawiki/2012/3/33/Ssumss1.PNG" width="200px"><br>
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<h7><ul><p dir="ltr"> This is a work in progress. We are currently working on the MoClo Levels 1 reactions and unfortunately we couldn`t get them ready before the Wiki freeze. Stay tuned for additional data during the World Jamboree.
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<li>Lastly, we sequenced the level 1 parts, but the results yielded sequences of only the destination vector and not the transcriptional unit. We are currently trouble shooting and we plan to present the results at the Jamboree.
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Latest revision as of 17:34, 26 October 2012

BostonU iGEM Team: Welcome



Results Summary



Creating Destination Vectors

    Below are the step by step results we obtained in making all of our destination vectors:

    First, we transformed the destination vectors into cells onto plates with IPTG/X-Gal for blue white screening. In this case, we wanted to see blue cells, indicative of the presence of LacZ. Our destination vectors also varied in whether the backbone is phosphorylated or dephosphorylated. In this picture, the dephosphorylated showed higher transformation efficiency by preventing recircularization of the backbone, yielding more cells transformed with the LacZ as part of the destination vector.



    We checked whether the destination vectors contained the LacZ fragment amplified with the moclo fusion sites by running digest of cut vs uncut. In the gel picture, each SM refers to a different destination vector and the C and UC means cut and uncut, respectively.



    The final step is sequencing the destination vectors to confirm the correct orientation of LacZ. In the nucleotide sequence file and the complementary diagram, the LacZ sequence, 2 fusion sites and restriction sites are highlighted in different colors.



    The trace file shows confidence of the sequencing.



    Converting Biobrick to Moclo Parts:

      We first added fusion sites to Biobrick parts through PCR. The primers contained the sequence for the fusion sites as part its overhang. In the process we used regular PCR amplification and also ligation PCR.

      The first picture refers to promoters R0010 and R0079 with the added fusion sites



      The second picture is the result of ligation PCR of the J series promoters.



    Creating Moclo Level 0 Parts

      Afterwards we transformed and plated cells with level 0 Moclo parts on to IPTG/X-Gal plates for blue white screening where we look for white colonies. In the picture we see white colonies, indicating the LacZ was cut out of the destination vector and replaced by parts with the Moclo fusion sites.



      We confirmed the Moclo level 0 parts through sequencing. Below there is an example of the sequence file we received for a promoter, RBS, gene and terminator. The files were edited with highlights to indicate the correct locations of the part, fusion sites and the restriction sites.








    Creating Level 1 Moclo Parts

      This is a work in progress. We are currently working on the MoClo Levels 1 reactions and unfortunately we couldn`t get them ready before the Wiki freeze. Stay tuned for additional data during the World Jamboree.




    Retrieved from "http://2012.igem.org/Team:BostonU/Results"