Difference between revisions of "Part:BBa K2671000"

Revision as of 07:36, 17 October 2018

AraC-pBAD-mNG

iGEM17_Linkoping_Sweden part BBa_K2474000 modified. A stop codon was mutated behind mNeonGreen (mNG). The original part was mNG fused to Aβ1-42, where the fusion protein had its stop codon after Aβ1-42. Another mutation was made in the GS-linker sequence where a SpeI-site was added and the sequence for Aβ1-42 was cleaved off. With these improvements mNG can now be used as a reporter in a fully functional operon with the pBAD-AraC inducible system. It also compares the difficulty an aggregation-prone fusion protein to a non fused version.

Usage and Biology

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 1. 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, 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 fig 1.

Figure 1. From left to right: Reference, mutated version (this biobrick) and the unmutated version (BBa_K2474000).

Sequencing

As seen in fig 1. the improved version has a much stronger color, which indicates that the Aß1-42 fusion was successfully terminated. And as seen in fig 2, 3. the sequencing results confirms this. File:T--Linkoping Sweden--BBa K2671000seq.zip Sequencing data files. The analyzed data in figure 2 and 3. is from these files. These contain full information on the sequencing including a chromatogram.

Figure 3. Comparing the sequencing results of the mutated version (this biobrick) and the template (BBa_K2474000). Showing a successful addition of an stop codon. Comparing the length of the sequence, both done with the VR primer, a loss of ~170 bases. This confirms a successful removal of the Aβ1-42-tail.

Figure 2. Sequencing results (see attached files for an chromatogram) analyzed in benchling, annotations added for clarity. The sequencing data attached confirms a successful removal of Aβ1-42 and addition of a stop codon. Sequencing was done with the VR primer.

Sequence and Features