Team:Oxford/biosensor construction
From 2014.igem.org
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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 electronic circuit to complement this genetic one: Production of the DCM-binding protein DcmR
![](https://static.igem.org/mediawiki/2014/b/b6/Oxford_pOXON-2-dcmR-mCherry_text.png)
The binding site for DcmR with expression-reporting GFP
![](https://static.igem.org/mediawiki/2014/1/12/Oxford_pSRK-Gm-pdcmAsfGFP_text.png)
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).
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 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’).
These are shown below:
![](https://static.igem.org/mediawiki/2014/5/52/Oxfordigem_pj404forward.png)
![](https://static.igem.org/mediawiki/2014/c/c4/Oxfordigem_pj404reverse.png)
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![](https://static.igem.org/mediawiki/2014/8/84/Oxford_Fran_flowchart1.png)
Building pOXON-1
Building pOXON-2 and pOXON-2-dcmR
Adding in mCherry
Building pSRK Gm construct
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