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. 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 electronic circuit to complement this genetic one:<br><br>
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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 sourced 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 electronic circuit to complement this genetic one:<br><br>
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Unfortunately after multiple attempts to construct this pSRK Gm pdcmAsfGFP construct we were unable to do so through Gibson assembly. Since we plan to prove this system can work in E. coli we were able to re-design this construct to use a different vector with a origin of replication compatible with our other construct pOXON-2 (containing dcmR).  
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Unfortunately, we were unable to assemble the pSRKGm pdcmAsfGFP construct even after multiple tries. Since we plan to prove that this system can work in E. coli, we re-designed this construct to use a different vector with an origin of replication that is compatible with our other construct pOXON-2 (containing dcmR).  
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Since DcmR is predicted to regulate expression of DcmA as well as auto regulation of its own expression we decided to insert this promoter-containing intergenic region with GFP at both positions. These positions correspond to the equivalent position of dcmA (labelled as ‘forward’) or the equivalent position of dcmR (labelled as ‘reverse’).  
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Since DcmR is predicted to regulate expression of DcmA as well as auto-regulating its own expression, we decided to insert this promoter-containing intergenic region with GFP at both positions. These positions correspond to the equivalent position of dcmA (labelled as ‘forward’) or the equivalent position of dcmR (labelled as ‘reverse’).  
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<li>pOXON-1 was produced using the Gibson assembly method.</li><br>
<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>
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<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>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>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>
<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>
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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 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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<li>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 sources other than the gblock. Firstly, pdcmA will be amplified from Methylobacterium extorquens DM4 genomic DNA and inserted into the pSRKGm vector. sfGFP will then be amplified from a plasmid already containing it, and added to the pSRKGm-pdcmA construct.</li><br>
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Revision as of 22:55, 13 October 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 sourced 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 electronic circuit to complement this genetic one:

pOXON-2-dcmR-mCherry -

Production of the DCM-binding protein DcmR

pOXON-2-dcmR-mCherry
Oxford iGEM 2014
pSRK-Gm-pdcmAsfGFP and pJ404-pdcmAsfGFP -

The binding site for DcmR with expression-reporting GFP

pSRK-Gm-pdcmAsfGFP




Unfortunately, we were unable to assemble the pSRKGm pdcmAsfGFP construct even after multiple tries. Since we plan to prove that this system can work in E. coli, we re-designed this construct to use a different vector with an origin of replication that is compatible with our other construct pOXON-2 (containing dcmR).

We chose to use plasmid backbone pJ404 since it contains a pBR322 origin of replication which is compatible with p15A origin of replication present in pOXON-2.

Since DcmR is predicted to regulate expression of DcmA as well as auto-regulating its own expression, we decided to insert this promoter-containing intergenic region with GFP at both positions. These positions correspond to the equivalent position of dcmA (labelled as ‘forward’) or the equivalent position of dcmR (labelled as ‘reverse’).

These are shown below:




Oxford iGEM 2014
Why these 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


  • 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 sources other than the gblock. Firstly, pdcmA will be amplified from Methylobacterium extorquens DM4 genomic DNA and inserted into the pSRKGm vector. sfGFP will then be amplified from a plasmid already containing it, and added to the pSRKGm-pdcmA construct.







    • Oxford iGEM 2014

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