Part:BBa_M50021:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_M50021

Our sensor and actuator construct (TS-Lim-P) was created through Golden Gate cloning and a Bsal restriction enzyme, which were used to insert this part into DNA 2.0's E. coli promoter cassette. 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

The GFP protein should not have been produced by bacteria containing this sequence, since the terminator was placed before the GFP gene in our plasmid construct (see plasmid map above. These samples, however, were included in the GFP assay conducted for BBa_M50018 to act as extra negative controls and to collect cell density measurements. However, they are clearly fluorescent. Furthermore, the increased fluorescence production rate at higher temperatures. 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. Because the GFP gene follows our d-limonene synthase gene, it is likely that both are being transcribed. It is unclear from these data, however, whether or not d-limonene synthase is adequately translated and functioning.

GFP production rates increased from 2.65 at 25°C to 3.66 at 37°C to 8.30 at 42ºC. This data demonstrates that there was a significant increase in rate of GFP production as temperature increased, which asserts our hypothesis that the σ32 promoter creates heat-sensitive protein expression. The fluorescence always increased linearly over time, which is supported by the high R2 values of our lines of best fit, all of which are near or above 0.9. This linear trend is further discussed in our conclusion. As expected, the E.Coli control had little fluorescence resulting in a relatively flat line of best fit, and indicating a GFP production rate close to zero.

GC-MS Limonene Detection

User Reviews

UNIQbe0d9d58172e27d6-partinfo-00000000-QINU UNIQbe0d9d58172e27d6-partinfo-00000001-QINU