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Team:Oxford/biosensor construction
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Construction
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 the following two plasmid systems:
Oxford iGEM 2014
Oxford iGEM 2014
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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.
• This was achieved by Gibson assembly. - 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 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. Firstly pdcmA will be inserted to make the construct pOXON-3, then sfGP will be added.
Building pOXON-1
• This was achieved by Gibson assembly. - 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 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. Firstly pdcmA will be inserted to make the construct pOXON-3, then sfGP will be added.
