Part:BBa_K2671000

AraC-pBAD-mNG

Liu iGEM2017s plasmid (BBa_K2474000) with an improvement. 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.

Usage and Biology

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

Verification 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. the sequencing results confirms this. Both BBa_K2671000 and BBa_K2474000 was grown for 16 hours in 37 degrees and induced from the start with 0.25 mg/ml L-arabinose. They were then lysed with the freeze-thaw method and centrifuged. After the supernatant was saved and placed on an UV table, see fig. 1.

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 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.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1267
  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961