Difference between revisions of "Part:BBa K2671000"
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<partinfo>BBa_K2671000 short</partinfo> | <partinfo>BBa_K2671000 short</partinfo> | ||
− | Liu iGEM2017s plasmid (BBa_K2474000) | + | 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=== | ===Usage and Biology=== | ||
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[[File:T--Linkoping_Sweden--improved.jpeg|450px|thumb|left|Figure 1. From left to right: Reference, mutated version (this biobrick) and the unmutated version (BBa_K2474000). ]] | [[File:T--Linkoping_Sweden--improved.jpeg|450px|thumb|left|Figure 1. From left to right: Reference, mutated version (this biobrick) and the unmutated version (BBa_K2474000). ]] | ||
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+ | <h2>Verification</h2> | ||
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. | 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. | ||
− | [[File:T--Linkoping_Sweden--chromatogram.png|450px|thumb| | + | [[File:T--Linkoping_Sweden--chromatogram.png|450px|thumb|center| 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. ]] |
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+ | [[File:T--Linkoping_Sweden--improveseq1.png|200px|thumb|center|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.]] | ||
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[[File:T--Linkoping Sweden--BBa K2671000seq.zip|200px|thumb|right|]] | [[File:T--Linkoping Sweden--BBa K2671000seq.zip|200px|thumb|right|]] | ||
Sequencing data files. The analyzed data in figure 2. is from these files. These contain full information on the sequencing including a chromatogram. | Sequencing data files. The analyzed data in figure 2. is from these files. These contain full information on the sequencing including a chromatogram. | ||
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Revision as of 06:45, 17 October 2018
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
Comparing
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.
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