Team:Oxford/biosensor construction

From 2014.igem.org

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<h1>Introduction: how we constructed our biosensor</h1>
<h1>Introduction: how we constructed our biosensor</h1>
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In order to be able to use our model and to determine whether DcmR acts as a repressor or activator in the presence of DCM we designed and constructed the following two plasmid system:
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In order to be able to use our model and to determine whether DcmR acts as a repressor or activator in the presence of DCM we designed and constructed the following two plasmid system. We primarily used Gibson assembly methods and source most of the necessary DNA from gblocks(synthesised oligonucleotides) we had designed based in the sequenced genome of Methylobacterium DM4. This system will also form the DCM biosensor and will be integrated with an elctronic circuit to complement this genetic one:
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<h1>Building pOXON-1</h1><br>
<h1>Building pOXON-1</h1><br>
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<li>The first task in the construction of the pOXON-2-dcmR-mcherry construct was the creation of pOXON-1; pME6010 with tetracycline resistance replaced by kanamycin resistance. The KanR gene was amplified with an optimised RBS.</li><br>
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<li>The first task in the construction of the pOXON-2-dcmR-mcherry construct was the creation of pOXON-1; pME6010 with tetracycline resistance replaced by kanamycin resistance. (N.B. The KanR gene was amplified with an optimised RBS.)</li><br>
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<li>This was achieved by Gibson assembly.</li><br>
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<li>pOXON-1 was produced using the Gibson assembly method.</li><br>
<h1>Building pOXON-2 and pOXON-2-dcmR</h1><br>
<h1>Building pOXON-2 and pOXON-2-dcmR</h1><br>
<li>pOXON-1 was then used as  the vector for the insertion of the three gblock  fragment constituting the inducible expression system of dcmR via Gibson assembly.</li><br>
<li>pOXON-1 was then used as  the vector for the insertion of the three gblock  fragment constituting the inducible expression system of dcmR via Gibson assembly.</li><br>
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<li>Upon sequencing of the product, it was determined that the version of the gblock containing the dcmR gene in the construct was actually truncated. This construct with the truncated dcmR is pOXON-2. A second Gibson assembly reaction was used to replace the truncated version with the full length gene also derived from the gblock. The resulting construct named pOXON-2-dcmR.</li><br>
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<li>Upon sequencing of the product, it was determined that the version of the gblock containing the dcmR gene in the construct was actually truncated. This construct with the truncated dcmR is pOXON-2. A second Gibson assembly reaction was used to replace the truncated version with the full length gene also derived from the gblock. The resulting construct was named pOXON-2-dcmR.</li><br>
<h1>Adding in mCherry</h1><br>
<h1>Adding in mCherry</h1><br>
<li>We then used pOXON-2-dcmR as the vector for the insertion of mCherry downstream of dcmR as a translational fusion by Gibson assembly.</li><br>
<li>We then used pOXON-2-dcmR as the vector for the insertion of mCherry downstream of dcmR as a translational fusion by Gibson assembly.</li><br>
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<li>All constructs were confirmed by sequencing.</li><br>
<li>All constructs were confirmed by sequencing.</li><br>
<h1>Building pSRK Gm construct</h1><br>
<h1>Building pSRK Gm construct</h1><br>
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<li>Still under construction, we have attempted to make our second construct by inserting the pdcmAsfGFP  gblock into the pSRK Gm vector by Gibson assembly. As this is proving difficult the next approach will be to insert the two components separately. Firstly pdcmA will be inserted to make the construct pOXON-3, then sfGP will be added.</li><br>
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<li>Still under construction, we have attempted to make our second construct by inserting the pdcmAsfGFP  gblock into the pSRK Gm vector by Gibson assembly. As this is proving difficult the next approach will be to insert the two components separately and to source the DNA from other sources instead of the gblock. Firstly pdcmA will be amplified from Methylobacterium extorquens DM4 genomic DNA and inserted into the pSRKGm vector. sfGFP will them be amplified from a plasmid already containing it and then added the the pSRKGm-pdcmA construct.</li><br>
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Revision as of 13:46, 24 September 2014


Construction


Introduction: how we constructed our biosensor

In order to be able to use our model and to determine whether DcmR acts as a repressor or activator in the presence of DCM we designed and constructed the following two plasmid system. We primarily used Gibson assembly methods and source most of the necessary DNA from gblocks(synthesised oligonucleotides) we had designed based in the sequenced genome of Methylobacterium DM4. This system will also form the DCM biosensor and will be integrated with an elctronic circuit to complement this genetic one:
pOXON-2-dcmR-mCherry
pOXON-2-dcmR-mCherry
Oxford iGEM 2014
pSRK-Gm-pdcmAsfGFP
pSRK-Gm-pdcmAsfGFP
Oxford iGEM 2014
Why these two plasmid backbones?
Why these two plasmid backbones?
  • The two plasmids are partitioned during cell division by different systems, thus an equal proportion of each plasmid is maintained in each new daughter cell.

  • Different antibiotic resistances will allow us to select for cells that have taken up both plasmids by application of both antibiotics.

  • The replication origins compatible with E.coli and pseudomonas strains.

  • We have used two plasmids so that we can test each part in isolation before transforming them both into the same cell.
    • How were the constructs made?
    How were the constructs made?

    Building pOXON-1


  • The first task in the construction of the pOXON-2-dcmR-mcherry construct was the creation of pOXON-1; pME6010 with tetracycline resistance replaced by kanamycin resistance. (N.B. The KanR gene was amplified with an optimised RBS.)

  • pOXON-1 was produced using the Gibson assembly method.

    • Building pOXON-2 and pOXON-2-dcmR


  • pOXON-1 was then used as the vector for the insertion of the three gblock fragment constituting the inducible expression system of dcmR via Gibson assembly.

  • Upon sequencing of the product, it was determined that the version of the gblock containing the dcmR gene in the construct was actually truncated. This construct with the truncated dcmR is pOXON-2. A second Gibson assembly reaction was used to replace the truncated version with the full length gene also derived from the gblock. The resulting construct was named pOXON-2-dcmR.

    • Adding in mCherry


  • We then used pOXON-2-dcmR as the vector for the insertion of mCherry downstream of dcmR as a translational fusion by Gibson assembly.

  • We therefore have a system of expressing dcmR with (pOXON-2-dcmR-mCherry) and without (pOXON-2-dcmR) the mCherry fusion in order to test whether the addition of mCherry affects the action of DcmR. Both will be submitted as BioBricks in the standard pSB1C3 backbone.

  • All constructs were confirmed by sequencing.

    • Building pSRK Gm construct


  • Still under construction, we have attempted to make our second construct by inserting the pdcmAsfGFP gblock into the pSRK Gm vector by Gibson assembly. As this is proving difficult the next approach will be to insert the two components separately and to source the DNA from other sources instead of the gblock. Firstly pdcmA will be amplified from Methylobacterium extorquens DM4 genomic DNA and inserted into the pSRKGm vector. sfGFP will them be amplified from a plasmid already containing it and then added the the pSRKGm-pdcmA construct.