Part:BBa_M50021:Experience
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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. 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. Final plasmid map shown here:
Heat Sensitivity Assay
These samples were only included in the first place to act as extra negative controls and to collect cell density measurements. The GFP protein should not have been produced by the bacteria, since the terminator was placed before the GFP gene in our plasmid construct (Fig.3). However, the high fluorescence and increased fluorescence production rate at higher temperatures indicated otherwise. Similar increases in GFP production rate between the two plasmid constructs further supported the realization that GFP was expressed in a heat-dependent manner in the TS-Lim-P cultures.
GC-MS Limonene Detection
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