Team:Exeter/Results/inducible

The 3-Gene Inducible Plasmid - Freddie Dudbridge

The three gene inducible plasmid would lead the way to our technological revolution by proving the principle that polysaccharides could be created on demand. To show this we would put three different genes encoding three different glycosyltransferases, behind different promoters. After modelling the plasmid using Tinkercell, it was found that repressible promoters would give better results than inducible promoters.

We found that there was not a large choice of promoters available. for our first gene , wbnJ, we chose a constitutive promoter BBa_J23119. The second gene, wbnK would be controlled by the repressible promoter BBa_J13002, which responds to tetracycline. The last gene in the plasmid, wbnK, would be controlled by the repressible promoter BBa_K094120 which responds to IPTG.

To construct our Plasmid we would use 3A assembly. There is a large amount of cloning in this device. 3A assembly does not require gel purification and can be selected on antibiotics. This technique would also allow the creation of the individual genes behind the promoters. The promoters could then be characterised and an assay run on the individual genes.

Sequencing was used to confirm our results after being screened on a gel (Fig. 1).

For those Promoter_RBS constructs that we sequenced, it was a clear cut sequence like below (Fig. 2).

We had to be careful with cross contamination due to mixed reads. Other issues could have been a low DNA concentration or the primer failing to anneal properly.

Promoter_RBS sequences constructed by 3A assembly:

• BBa_K094120_BBa_B0034
• BBa_K206000_BBa_B0034
• BBa_K206001_BBa_B0034
• BBa_I0500_BBa_B0034
• BBa_J23119_BBa_B0034

Genes cloned using 3A assembly:

• Wbnk_BBaB0014
• WclY_BBaB0014
• WfcA_BBaB0014
• WbiP_BBaB0014
• WbbC_BBaB0014

Devices made:

• BBa_K094120_BBa_B0034_ WclY_BBaB0014
• BBa_J13002_ Wbnk_BBaB0014

Two gene plasmid made:

• BBa_J13002_ Wbnk_BBaB0014_ BBa_K094120_BBa_B0034_ WclY_BBaB0014
• BBa_J23119_BBa_B0034 and WbnJ_BBaB0014 needs to be added infront of this device for the construct to be achieved.

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