With the use of site-direted mutagenesis we wanted to improve the part of last years LiU iGEM team (BBa_K2474000). This was done by removing the fusion protein and the unnecessary bases as shown in figure 1. By doing this the fluorescent protein mNeonGreen (mNG) can be used as a reporter in a fully functional operone, with the use of the AraC/pBAD inducible system. The two additions was made with site-directed mutagenesis. Firstly the stop codon was added and sequenced, which was later used as the new template for the mutation to add the SpeI site. As seen in figure 1, we used SpeI to cleave off the unnecessary bases from the plasmid, and later ligated it back together without the amyloid-beta 1-42 fragment.
As seen in figure 2, our mutagenesis had a higher intensity on a UV-table.
Figure 1. A illutration of the steps made.
Results
Figure 2. From left to right: Reference, BBa_K2671000 (mutated part) and BBa_K2474000.
