Team:Wisconsin-Madison/SDF

• Team

  • Team Members
  • Advisors
  • Sponsors

• Projects

  • Engineering Limonene Production in E.coli
  • Translational Coupling Cassette (TCC)

• Parts

  • Translational Coupling Cassette
  • Negative TCC Control
  • Positive TCC Control
  • Codon-optomized LIMS1

• Modeling

• Protocol

• Safety

• Human practice

How the translational coupling cassette works

The translational coupling cassette is designed to determine if a target gene is being fully translated. This is achieved by fusing a DNA sequence to the 5’ end of the open reading frame that forms an RNA hairpin structure following transcription. Embedded in this mRNA hairpin is a His-tag and a stop codon for the target gene followed by a ribosome binding site for a downstream reporter gene. In our construct, the reporter gene is a red fluorescent protein (RFP) from the parts registry (BBa_E1010). If the upstream gene of interest is not being properly translated, the hairpin will remain intact and block the translation of the RFP reporter gene by occluding the RBS (Figure 1). However, if the 5’ gene of interest is being properly translated, the engineered hairpin is designed so that ribosomal helicase activity is sufficient to break the hairpin apart, enabling both complete translation of the gene of the target gene and allows access to the RBS of the reporter gene (Figure 2). This permits loading of a ribosome and translation of the reporter gene. This system allows a user to indirectly determine if their gene of interest is being fully translated; complete translation will yield a detectable amount of RFP – the reporter gene.

Assays for the translational coupling cassette

Two primary assays used to characterize the fluorescence produced by the RFP reporter from the translational coupling cassette (TCC).The first was performed using a Typhoon Imager (GE Healthcare). This instrument excites a sample with a laser (532 nm) and detects the emitted fluorescent signal via a photomultiplier tube (PMT). We spotted droplets of our various strains on selective media plates which contained exactly 20mL of agar solution. The droplets were absorbed into the agar and the plates were incubated overnight before being imaged on the Typhoon Imager. Below is a list of the different constructs we imaged using this method.

Construct	Purpose
TCC	Negative control for fluorescence
J23102-E0040-TCC	Positive control – test for translation of the target gene without the use of the TCC
J23102-Limonene synthase(Lims1)-TCC	Testing translation
J23102-Lims1-Stop codon(Stop)-TCC	Negative control for testing translation
J23102-Codon optimized(CO)-Lims1-TCC	Testing translation
J23102-CO-Lims1-Stop-TCC	Negative control for test of translation
J23102-E0840	Testing the amount of RFP signal and ensuring that we account for GFP fluorescence bleed-through into the RFP channel in our positive control construct

J23102 is a constitutive promoter; TCC stands for translational coupling cassette; E0040 is a GFP open reading frame; E0840 is a composite part containing an ribosome binding site, E0040 ORF, and a terminator.

The J23102+E0040+TCC was used as a positive control to test the functionality of the translational coupling cassette. We used a green fluorescent protein (E0040) as our positive control because it was ideal to see if we were getting translation of a target gene without the use of the coupling cassette. Additionally, this part is well characterized and routinely used by many iGEM teams, suggesting it should not have any issues being fully translated. However, there was one drawback to using the GFP as our target gene; when scanning our samples in the Typhoon Imager, we noticed that the GFP only construct (J23100 + E0040) produced low levels of fluorescence intensity 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 really from the RFP reporter gene or was just bleed-through from the GFP. Figure below demonstrates that the signal intensity from the GFP-TCC-RFP construct is well above the signal produced from the GFP only strain, suggesting that our translational coupling cassette is working as intended.

Figure 4: Plot of fluorescence over turbidity

Figure 5: Plot of fluorescence over turbidity

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. Second, the construct containing a stop codon midway through the target gene is not generating RFP. This demonstrates that the hairpin stays intact when successful translation of the target gene does not occur. Third, the codon-optimized limonene synthase is being translated, and the non-codon optimized is not. The fluorescence produced by the codon-optimized construct is much lower than the positive control, but we cannot definitively explain this.

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.

This plot supports the conclusions drawn from the Typhoon images. The translational coupling cassette produces RFP. The codon optimized limonene synthase seems to be translating more efficiently than the non-codon optimized version. In fact, the limonene synthase may not be translating at all. However, we still have not been able to detect any amount of limonene in our GC/MS assays. We were able to make amorphadiene, so we know that the mevalonate pathway works correctly up until the GPP synthase gene. From this point onwards, all we know is that the limonene synthase (now codon optimized) is being translated. From here, we would require more time and materials to fully troubleshoot why no limonene is being produced. We do have a few hypotheses, based on observations and information about this pathway. First, limonene synthase may be forming an inclusion body in the cell. Second, it is a possibility that GPP is not being produced, and we would require standards (which are quite expensive) and a new protocol to detect for it on the GC/MS. If we had these standards, we would also be able to try to purify limonene synthase using our TCC his-tagged version. This purified enzyme could then be doped with GPP, and hopefully create limonene. If this was the case, we would know that GPP is not being produced, or something is happening to the limonene synthase between translation and the active synthesis of limonene. Third, many of the molecules and enzymes in the mevalonate pathway can be toxic to Escherichia coli cells at high levels, including limonene.

How to clone using the Translational Coupling Cassette

In order to use the translational coupling cassette, the target gene must be PCR amplified using BioFusion primers. If the gene is amplified using standard BioBrick primers, an open reading frame shift will occur, a premature stop codon will be formed, and the hairpin may not break. In addition to using BioFusion primers, the stop codon must also be taken out of the target gene being amplified for the same reason. If a stop codon prematurely stops translation, then the hairpin may not break and the response gene (RFP) will not be translated. As an example:

We cloned in the E0040 gene (GFP) into the TCC as the target gene. The first and last 30 base pairs of the E0840 ribosome binding site plus open reading frame (GFP) are as follows:

Reference: