Team:BostonU/Results

From 2012.igem.org

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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. <li>
<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. <li>
<br><img src="https://static.igem.org/mediawiki/2012/e/e3/Sum1.png" width="300px">
<br><img src="https://static.igem.org/mediawiki/2012/e/e3/Sum1.png" width="300px">
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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. The trace file shows confidence of the sequencing.<li>
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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. The trace file shows confidence of the sequencing.<li><img src="https://static.igem.org/mediawiki/2012/thumb/d/da/Sum3.png/800px-Sum3.png" width="500px"><ul><li>
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<img src="https://static.igem.org/mediawiki/2012/a/ac/Sum2.png" width="300px">
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<img src="https://static.igem.org/mediawiki/2012/0/04/Sum4.png" width="300px"><br>
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<h9>Converting Biobrick to Moclo Parts:<h9>
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<h7>
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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/thumb/d/da/Sum3.png/800px-Sum3.png" width="300px">seq veri dv1<br>
 
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<img src="https://static.igem.org/mediawiki/2012/0/04/Sum4.png" width="300px"> seq veri dv1 finch<br>
 
<img src="https://static.igem.org/mediawiki/2012/5/58/Sum5.png" width="300px">reg pcr<br>
<img src="https://static.igem.org/mediawiki/2012/5/58/Sum5.png" width="300px">reg pcr<br>
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<img src="https://static.igem.org/mediawiki/2012/6/6a/Sum7.png" width="300px">moclo 0 comp<br>
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<li>The second picture is the result of ligation PCR of the J series promoters.<br>
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<img src="https://static.igem.org/mediawiki/2012/9/98/Sum8.png" width="300px">ligationpcr<br>
<img src="https://static.igem.org/mediawiki/2012/9/98/Sum8.png" width="300px">ligationpcr<br>
<ul>
<ul>
<h9>MoClo level 0 Vectors and Parts</h9>
<h9>MoClo level 0 Vectors and Parts</h9>
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<img src="https://static.igem.org/mediawiki/2012/6/6a/Sum7.png" width="300px">moclo 0 comp<br>
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<p>
<h7>Things to include: gels, sequence data, plate pictures</h7>
<h7>Things to include: gels, sequence data, plate pictures</h7>

Revision as of 16:58, 3 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:

  • 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.
  • 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 reg pcr
    • The second picture is the result of ligation PCR of the J series promoters.
      ligationpcr
        MoClo level 0 Vectors and Parts moclo 0 comp

        Things to include: gels, sequence data, plate pictures level 1 moclo l0 moclo gene l0 moclo term l0 moclo rbs l0 moclo promotor


      Basic Genetic Circuits

        Things to include: SBOL figures of circuits, gels, sequence data, plate pictures, characterization data summary

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