Part:BBa_K4205111:Experience

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Since the 3D structure of LldR in our chassis, Escherichia coli Nissle 1917, was not found in PDB, we used SWISS-MODEL for homology modeling to predict the 3D structure of LldR.

Predicted 3D structure of LldR

With the help of AutoDock, we simulated the docking of LldR and L-lactate. The resulting figure is presented below. The docking sites are colored yellow.

Docking of LldR and L-lactate

We constructed the ALPaGA-lacZ plasmid and transformed it into Escherichia coli DH5α. To prove the success of our transformation, we designed primers, performed plasmid PCR and colony PCR to amplify the LldR gene, and ran the agarose gal.

Proof of the successful transformation.(A) plasmid PCR. Lanes 1-2: PCR sample form transformed DH5α. Lanes 3-4: PCR sample form untransformed DH5α. (B) colony PCR. Lanes 1-4: PCR sample form transformed DH5α. Lanes 5-8: PCR sample form untransformed DH5α.

As shown in the figure above, after plasmid PCR and colony PCR, the sample from the transformed DH5α both ran out bands near 750 bp, which was consistent with the target band (777 bp). This further indicated that our plasmid transformation was successful.