Team:SDU-Denmark/Tour44

Flavor improvement

The smell of E. coli as we know it from working in the lab using LB media, is not delicious. The first thought that comes to mind is not how much you would like to taste it - so we wanted to make E. coli taste and smell delicious. In order for us to do so, we wanted to make a limonene construct synthesizing limonene controlled by pTet. In iGEM parts registry, two parts encoding the limonene synthase 1 exist. We decided to use both parts and thus make two constructs in order to test which one is the most useful and has a more enhanced production of limonene. The parts are to be found as Bba_K118024 and Bba_I742110. Bba_K118024 encodes appY and dxs – Bba_I742110 does not. appY and dxs increases the production of limonene, so we expect Bba_K118024 to show a more enhanced production of limonene than Bba_I742110. Source: Kang, M. J., et al.: Identification of genes affecting lycopene accumulation in Escherichia coli using a shot-gun method. Biotechnology and Bioengineering, 2005. Vol. 91: 5, p. 636-642. (Link)

In addition to the limonene synthesis, we acquired an odor-free E. coli YYC912 strain from the Coli Genetic Stock Center. This strain differs from the wild type E. coli K12 MG1655 in that it does not produce indole. Our goal was to examine the smell of the odor-free strain compared to the wild type. That way we would determine which strain would be best suited for our Edible coli project. We also wished to clone the limonene constructs into the odor-free strain, to investigate if the lemon smell is more prevalent than in the wild type. Though due to time restraints this was not accomplished.

This comparison should show if there was a difference in the odor of the limonene synthesizing strains and the ones that did not produce limonene, and if the odor was improved. The way we wanted to test this, was to set up a blind test using overnight cultures of E. coli K12 wild-type, odor-free E. coli and the two limonene constructs in wild-type E. coli K12 (since transformation of limonene constructs into the odor-free strain did not succeed). The bacteria were supposed to be grown in minimal media, but since we received the sequencing data, saying that the ligations were not successful, we did not carry out the experiment.

We decided to investigate the difference between the odor-free and wild type strain. Because the focus of interest is on the difference in odor, we decided to analyze the two strains with Ion-Mobility Spectrometry (IMS). Since IMS analyzes molecules in the gas phase, this analytical technique is particularly suitable for our investigations. In order to minimize the effect of the media, M9 Minimal media was used to grow the two strains.

Figure 1: IMS spectrum of wild type E. coli K12 MG1655.

Figure 2: IMS spectrum of odor-free strain YYC912.

The graphs in figure 1 and 2 shows the results from the IMS analysis. Along the horizontal axis is the reciprocal of the reduced mobility (K₀^-1), along the vertical axis is intensity and along the axis going towards and away from the viewer is the retention time. At first sight it can be seen that the two spectra are somewhat different from each other, which substantiate that the two strains are different. The difference between the odor-free strain and wild type is, as mentioned, that the odor-free strain should not produce indole. Source: iGEM, 2006: Registry of Standard Biological Parts, (Link) We therefor wished to investigate whether the indole peak was present for the wild type and not the odor free. The indole in E. coli has earlier been found at K₀^-1=0.556 s V cm^-2. Source: Ochoa, M. L., et. al.: Characterization and Differentiation of Bacteria Using In Situ Derivatization Ion Mobility Spectrometry of Whole Cells and Chemometric Modeling, Ohio University, 2004, p. 25 (Link) In figure 3 the spectra is shown for wild type (WT), odor-free (OF) and air. As can be seen from the spectra there is a peak for wild type at 0.556 that is more intense than the two others. This indicates the indole produced by wild type E. coli. The spectra for the odor-free strain can be seen to follow the spectra for air. This shows that indole is not produced by the odor-free strain.

Figure 3: IMS spectrum of wild type, odor-free, and air at RT=3.