Part:BBa_K762000:Experience

Applications of BBa_K762000

The fluorescence produced by the translational coupling cassette was assessed using a Typhoon Imager (GE Healthcare, provided by the UW-Madison Biotech Center). This instrument excites a sample with a laser (532 nm) and detects the emitted fluorescent signal via a photomultiplier tube. We spotted 10 µL droplets of our various strains (listed below) on selective media plates containing exactly 20mL of LB agar. The plates were incubated overnight before being imaged on the Typhoon Imager and analyzed.

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 (BBa_K762002)

J23102-Limonene synthase (LIMS1)-TCC - Testing translation

J23102-LIMS1-Stop codon (stop)-TCC - Negative control for translation (BBa_K762001)

J23102-Codon optimized-LIMS1-TCC - Testing translation

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 J23102-E0040-TCC construct was used as a positive control to test the functionality of the translational coupling cassette; E0040 is well-characterized and routinely used by many iGEM teams, suggesting it should not have any issues being fully translated. When scanning samples in the Typhoon Imager, the GFP-expressing (J23100-E0040) construct that was included in the assay produced low levels of fluorescence in the red fluorescent channel. This is not an uncommon problem when using fluorophores, particularly fluorescent proteins, and is due to bleed-through of the GFP fluorescent signal into the RFP emission channel. Thus, some of the red fluorescence signal produced by our positive control construct (J23102-E0040-TCC) was due to the GFP. This required us to account for the bleed-through to determine if the RFP signal from our GFP-TCC-RFP construct was actually from the RFP reporter gene or just bleed-through from the GFP. Figure 3c. (below) demonstrates that the signal intensity from the GFP-TCC-RFP construct is well above the signal produced from the GFP-only strain, showing that our translational coupling cassette is working as intended.

Key for Figure A and B

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

The codon optimized limonene synthase, with a stop codon located in the middle of the gene, was used as our negative control for the cassette. If translation is stopped partway through the target gene, the helicase activity on the ribosome will not break the hairpin and RFP will not be generated.

From this data, three things can be determined. First, the positive control is producing RFP, meaning the hairpin is being broken by the ribosome. This is seen in Figure 3A and 3C, where the J23102-E0040-TCC is well above the negative controls. Second, the construct containing a stop codon midway through the target gene is not generating RFP, which is seen in the same two figures. This demonstrates that the hairpin stays intact when the target gene does not translate.

The second assay used to quantify fluorescence was done by using a 96-well plate reader. Optical densities and fluorescence were taken over a 24 period, and used to determine if our target gene was being translated. The same strains used in the Typhoon were tested using this method. Fluorescence was divided by optical density because all of the strains did not grow at the same rate. This allowed the normalization of fluorescence, and thus the quantification and comparison of it between strains.

These plots back up the conclusions drawn from the Typhoon images. The translational coupling cassette produces RFP and functions as expected.

The following link is a downloadable excel file containing the data from the 96-well plate reader, if you wish to compare any constructs you create to ours:

http://dl.dropbox.com/u/27094227/TCC-RFP-GFP%209-25-12%20CBJ4_final.xlsx

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