Experiments

Here you will find our protocols, which cover bacterial expression to DNA assembly of our siderophore, as well as troubleshooting steps. Each protocol contains all the materials needed for the experiment. In addition, all steps are described so that future iGEM participants can effectively build on our work.

In order to optimize the amount of DNA for our gBlock 3, the modifications made to the standard PCR protocol are already described in the gBlock amplification protocol (PCR amplification) with the necessary modifications to carry out the experiment. If you want to know more about this particular step, don’t forget to look into our Results page!

The vector assembly, pBluescript SK- with our 3 gBlocks, was assembled based on the manufacturer’s (NEB) Gibson Assembly® Protocol (E5510) | NEB.

We used the SOC medium (Super Optimal broth with Catabolite repression) to optimize the recovery of our Escherichia coli competent cells following transformation. In the end, our mutant selection was effectively done on LB agar plates with ampicillin, supplemented with X-gal, which allowed us to do a blue-white selection (Gibson assembly & transformation - Chemical transformation of competent cells). The cells that integrated our vector appeared white.