Improvement of Liu iGEM 2017s part BBa_K2474000

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 mutated version had a higher intensity on a UV-table.

Figure 1. A illutration of the steps made.

Results

The results for our improved part can be seen in below. The reason we choose to improve this part was not only to create a functional reporter system, but also to illustrate the loss of exchange with a aggregation prone fusion tag. From fig 2. you can clearly see that the version without amyloid-beta 1-42 folds better and thus gives a much higher fluorescence, while the fusion version is having trouble. The two parts was grown in LB-medium and 0.25 mg/ml L-arabinose over 16 hours. They were then lysed through the "freeze-thaw" method and centrifuged. The supernatant was saved and placed on an UV table as seen in fig 2.

Figure 2. From left to right: Reference with only water, BBa_K2671000 (mutated part) and BBa_K2474000.

Figure 2 shows that the mutated biobrick BBa_K2671000 has an increased intensity and could potentially be used as an effective operon. While the part BBa_K2474000 that is still linked to amyloid-beta has a lower intensity and aslo could not be used as a widely used operon.

Seen below in fig 3. is the chromatograms from sequencing. The results shows that the mutation GCC --> TTA was successful. The length of the sequence also suggest that amyloid-beta 1-42 was removed. Both parts was sequenced using the VR primer, and ~170 bases shorter fits completely with the length of the removed fragment.

Figure 3. The chromatograms from sequencing. Left is BBa_K2671000 (mutated part) and right is BBa_K2474000 (template).