Part:BBa_K762100:Experience

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Applications of BBa_K762100

For background on the assays for the production of limonene and information the associated precursor molecules, please see our wiki page: http://2012.igem.org/Team:Wisconsin-Madison/lemon

After trying several different methods for the in vivo production of limonene using E. coli host strains we were unable to detect limonene. We hypothesized that a lack of fully translated limonene synthase was the culprit. The limonene synthase gene is not native to E. coli, which can cause problems with translation due to differing codon usage. To remedy this, we created a codon optimized limonene synthase and repeated the initial methods of limonene production. Again, we were unable to detect limonene. To test our initial hypothesis, we adopted a translational coupling cassette to determine if the limonene synthase gene was being fully translated.

-Please see part page BBa_K762000 for an explanation on how the tranlational coupling cassette works (https://parts.igem.org/Part:BBa_K762000)-

Key for the following graphs:

TCC - Negative control for fluorescence

J23102-E0040-TCC - Positive control for the TCC construct, used to ensure the target gene was being translated without the need for the TCC

J23102-Limonene synthase (LIMS1)-TCC - Testing translation of part BBa_I742111

J23102-LIMS1-Stop codon (stop)-TCC - Negative control for translation

J23102-Codon optimized-LIMS1-TCC - Testing translation of part BBa_K762100

J23102-Codon optimized-LIMS1-Stop-TCC - Negative control for translation

J23102-E0840 - Testing the amount of RFP signal and ensuring we account for GFP fluorescence bleed-through into the RFP channel in our positive control construct.

The following graph was made from data from a 96-well plate assay and shows that limonene synthase (BBa_I732111) is not being translated, but our codon optimized version (BBa_K762100) is.

The following image is from a Typhoon Imager, and agrees with the data from the 96-well plate reader; codon-optimized limonene synthase is being translated, and non-codon optimized is not. (NS means no stop codon, i.e. the hairpin should be broken in the TCC).

Key for the Typhoon Images:

1 - GFP-TCC-RFP

2 - TCC only

3 - LIMS1-TCC

4 - LIMS1-Stop-TCC

5 - CO-LIMS1-TCC

6 - CO-LIMS1-Stop-TCC

Figure 3A: Quantification of fluorescence from Fig.3C by pixel saturation. From liquid cultures of each strain, three 10uL droplets were spotted onto 20mL LB agar plates with proper selection. Using Adobe Photoshop, the average pixel saturation of the three droplets from the Typhoon Image (Fig.3C) was calculated, normalizing for background fluorescence.Figure 3B: Plate as imaged by Nikon camera Figure 3C: Typhoon Image of plate in Fig.3B, edited using Adobe Photoshop

According to both sets of data, the codon optimized limonene synthase is being translated, and the non-codon optimized is not. Thus, we have improved the classic limonene synthase part (BBa_I742111). Unfortunately, this did not correspond to production of limonene. We must further troubleshoot to find out why.

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