With the use of site-directed mutagenesis the goal was to improve the part of last years LiU iGEM team biobrick BBa_K2474000 and make it to a reporter system which could be more widely used. The improved biobrick made this year (BBa_K2671000) in contrast to BBa_K2474000 could also be used to display how aggregation prone proteins affect fused reporters. This was done by removing amyloid-beta 1-42 via adding a restiction site (SpeI) and removing unnecessary bases as shown in figure 1. By doing this the fluorescent protein mNeonGreen (mNG) can be used as an efficient reporter gene with a different protein, instead of amyloid-beta 1-42. The promoter of this system is AraC/pBAD system [1].
The two additions were 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, SpeI was used to cleave off the unnecessary bases from the plasmid, and later ligated it back together without the amyloid-beta 1-42 fragment.
Figure 1. A illutration of the steps made.
Results
The results for our improved part can be seen in the figure below. The reason we chose to improve BBa_K2474000, as previously mentioned, was mainly to create a functional reporter system. However, this new part (BBa_K2671000) is able to illustrate how the loss of an aggregation prone fusion tag increase flouresence intensity. From Figure 2 it is visible that the new version without amyloid-beta 1-42 fused has an increased intensity when compared to the unmutated part.
The two parts were grown in LB-medium, 0.25 mg/ml L-arabinose and 25 ug/ml chloramphenicol 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 Figure 2
Figure 2. From left to right: Reference with only water, BBa_K2671000 (new improved mutated part made by LiU iGEM 2018) and BBa_K2474000 (unmutated part made by LiU iGEM 2017).
Sequencing
Seen below in Figure 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. The full sequencing files including chromatograms can be found at BBa_K2671000.
Figure 3. The chromatograms from sequencing. Left is BBa_K2671000 (mutated part) and right is BBa_K2474000 (template).
References
[1] Part:BBa K2474000 - parts.igem.org [Internet]. [cited 2018 Oct 18]. Available from: http://parts.igem.org/Part:BBa_K2474000
