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 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

We then created new samples of our TS-Lim-P cultures, now no longer focusing on the GFP activity, but rather quantifying limonene production. After taking out our samples from overnight incubation, there was often a noticeably sweet scent in our TS-Lim-P cultures, compared to our S-32-P cultures and E. coli controls. However, this scent would often dissipate very quickly after opening our test tubes, suggesting that the scent was caused by a volatile compound, which limonene is. In order to determine whether this odor was in fact limonene, we wanted to test for the presence of the compound using GC-MS. Following a previous iGEM team’s instruction on a successful extraction method followed by use of GC-MS to detect limonene, we layered 10 mL of dodecane over 40 mL of identical cell cultures. The dodecane overlay is used to trap the volatile limonene, ensuring that as much of the sample as possible is collected. We created six identical 40 mL experimental cultures, and layered half with dodecane. The samples without dodecane served to confirm that dodecane did not inhibit cell growth, and act as backups in case it did. One sample with and one without dodecane were incubated at each temperature (25°C, 37°C and 42°C) overnight for eighteen hours. We also included two flasks of untransformed E. coli, one with and one without dodecane at the standard 37ºC as negative controls. Resource and time limitations prevented us from including a negative control at each temperature, which would have been ideal.

This mass spectrometry data is taken from the sharp GC abundance peak at ~6.6 minutes, which proves this to be limonene

We prepared several limonene standards to run through the GC-MS to ensure the limonene was detectable. We made four dilutions of the standard at 100 mg/L, 50 mg/L, 25 mg/L, and 1 mg/L using ethyl acetate as a solvent. The detection levels of the standards would be used to create an algorithm allowing us to calculate our limonene yield. After the first GC-MS run, we found that the limonene standard eluted at ~6.5 minutes and displayed a clear peak at 136 g/mol, the molar mass of limonene. We ran our samples at 60°C for two minutes, which then increased at 10ºC per minute to 120°C, which was then held for another two minutes. Sample size was 1 µL with a helium flow rate of 1 mL/min and 1:10 split ratio.

We then prepared our cultures to run through the GC-MS. Since we did not how much limonene, if any, would be in the dodecane layer and did not want to oversaturate the machine, we made 100x, 50x, 10x and 1x dilutions using ethyl acetate as our solvent. After running our 100x sample, we found no detectable presence of limonene and decided we needed to drastically increase our concentration to get any sort of result. We moved on to testing our undiluted 1x sample, and unfortunately, there still were no detectable signs of limonene at any incubation temperatures:

Data is not shown, but while the standard's sharp peak at 6.6 minutes is that of limonene, the mass spec analysis from the slight GC peaks from the E. coli curves at this elution time do not match that of limonene

Stanford Location

Plasmid name: TS-Lim-P

DNA 2.0 gene #: 273935

Organism: E. coli

Sensor & Actuator

Barcode #s: 0133027126, 0133023878, 0133021782

Box label: BioE44 F16