Difference between revisions of "Part:BBa K2671000"

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<partinfo>BBa_K2671000 short</partinfo>
 
<partinfo>BBa_K2671000 short</partinfo>
  
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Liu iGEM2017s plasmid (BBa_K2474000) with an improvement. A stop codon was mutated behind mNeonGreen (mNG). The original part was mNG fused to amyloid-&#946;, where the fusion protein had its stop codon after amyloid-&#946;. Another mutation was made in the GS-linker sequence where a SpeI-site was added and the sequence for amyloid-β 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.
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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===
 
===Usage and Biology===
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Verification
 
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.
 
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.
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[[File:T--Linkoping_Sweden--improveseq1.png|200px|thumb|left|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.]]
  
 
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Revision as of 14:46, 15 October 2018


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.

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