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.

Figure 2. Sequencing results analyzed in benchling, annotations added for clarity. The sequencing data verifies 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