Difference between revisions of "Part:BBa M50021:Experience"
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===Applications of BBa_M50021=== | ===Applications of BBa_M50021=== | ||
| + | Our sensor and actuator construct (TS-Lim-P) was created through Golden Gate cloning, so it consisted of a BsaI restriction site followed by the repeated G sequence, the σ32 sequence, a strong RBS, the d-Limonene synthase gene with terminator, the repeated A sequence, and a second BsaI restriction site (Fig. 3) A BsaI restriction enzyme was used to insert our TS-Lim-P construct into the cassette with no extraneous bases. The terminator at the end of the d-limonene synthase was intended to prevent GFP from also being transcribed, as restrictions on cost and enzyme capabilities left us unable to remove this gene altogether. We derived the limonene synthase gene from the BioBrick part BBa_K1660003, which included a sequence already optimized for transcription in E. coli. The original BioBrick included a promoter sequence and limonene synthase sequence, but we only used the limonene synthase portion of the BioBrick. This d-limonene synthase codon sequence in the brick has been changed from the official sequence for d-limonene synthase in Citrus unshiu found on the NCBI website, but the amino acids encoded for are all equivalent. These changes optimize G/C content and codon abundance for production in E. coli. | ||
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| + | [[File:TS-Lim-P plasmid map V2.jpeg|400px|thumb|center]] | ||
===User Reviews=== | ===User Reviews=== | ||
Revision as of 03:05, 12 December 2016
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Applications of BBa_M50021
Our sensor and actuator construct (TS-Lim-P) was created through Golden Gate cloning, so it consisted of a BsaI restriction site followed by the repeated G sequence, the σ32 sequence, a strong RBS, the d-Limonene synthase gene with terminator, the repeated A sequence, and a second BsaI restriction site (Fig. 3) A BsaI restriction enzyme was used to insert our TS-Lim-P construct into the cassette with no extraneous bases. The terminator at the end of the d-limonene synthase was intended to prevent GFP from also being transcribed, as restrictions on cost and enzyme capabilities left us unable to remove this gene altogether. We derived the limonene synthase gene from the BioBrick part BBa_K1660003, which included a sequence already optimized for transcription in E. coli. The original BioBrick included a promoter sequence and limonene synthase sequence, but we only used the limonene synthase portion of the BioBrick. This d-limonene synthase codon sequence in the brick has been changed from the official sequence for d-limonene synthase in Citrus unshiu found on the NCBI website, but the amino acids encoded for are all equivalent. These changes optimize G/C content and codon abundance for production in E. coli.
User Reviews
UNIQ600039bf96f7fab3-partinfo-00000000-QINU UNIQ600039bf96f7fab3-partinfo-00000001-QINU