Part:BBa_K2671000
AraC-pBAD-mNG
Liu iGEM2017s plasmid (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 a 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 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 1.
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. is from these files. These contain full information on the sequencing including a chromatogram.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1267
Illegal BamHI site found at 1144 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
None |