Team:Edinburgh/Project/Future

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Sequential Addition

One of the future expansions of BRIDGE which we have discussed is using it to directly introduce genes in the genome next to each other without using the BioBrick method before-hand. If you wanted to insert 4 genes with the steps described above, it would take 8 steps. If you do this with the method below it would take 4 steps.

At the first step of the process, the first antibiotic resistance and sacB are introduced alongside the first gene. The antibiotic resistance gene can then be replaced with the next gene and a second antibiotic resistance gene, thereby cycling the antibiotic resistance such that selection is different at each step. At the last step, both markers are removed and the final constructs can be selected for by growth on sucrose (growth on sucrose can also be used as a negative control at each stage).

The final construct would look as below:

References