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From 2014.igem.org

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Chromoproteins

As an example output plugin we transformed into Marchantia a selection of the chromoproteins brought to iGEM by Uppsala 2011. This was to test whether a simple change in colour would work as an easily visible reporter.

Five chromoproteins were selected for expression: eforred (BBa_K592012), tspurple (BBa_K1033906), aspink (BBa_K1033927), aeblue (BBa_K1033929) and amilCP(BBa_K1033930, a deep blue colour). The N7 nuclear localisation tag (INSERT BIOBRICK NUMBER HERE) was added to tspurple, aspink and amilCP. Each was put into pGreen

The transformation procedure yielded a large number of transformants, approximately 100 per construct, which were confirmed via PCR using homogenised plants as a template and chromoprotein-specific primers for each tDNA added.

Left: A PCR of the transformed plants showing successful transformation. Right: A plate of transformants growing happily on hygromycin

For each chromoprotein construct, the transformation yielded approximately 100 transformed sporelings (7-days old plants). The petri dish to the right contains hygromycin as a selective marker. The gel confirms the transformation in three plants each from two other plates. The forward and reverse primers used in the PCR anneal to 35S and N7 respectively, and the template was homogenised plant material.

After approximately four weeks of growth, sparsely distributed bright red cells became apparent under the microscope in the plants transformed with the tsPurple gene. Although similar cells were not found in wild type plants, diffuse red regions were found and are probably due to release of anthocyanins by stressed or aging cells.

The bright red cells seen in Marchantia transformed with the TsPurple construct

It is notable that in the red cells, the vacuole is not visually distinct from the cytoplasm. As GFP, a protein of similar structure, is not accumulated in the vacuole without specific targeting, this implies that either the colour we observe in the bright cells is not due to chromoprotein expression, or perhaps that tsPurple is toxic enough to cause cellular damage and leaky organelles.

Plants transformed with asPink (which is also known as asFP595 due to its fluorescence), nuclear localised with an N7 tag, while not visibly pigmented, did have areas that fluoresced at the literature wavelengths.

Part of an asPink:N7 transformant visualised using a fluorescence microscope with a GFP filter (the only one with wavelength windows that intersect those of asPink's absorption and emission peaks)

Raspberry ketone production

As another example output module, the biosynthetic pathway from coumaroyl-coA to raspberry ketone was assembled, to allow the smell of raspberries to be used as an odour signal that the input module has been activated.

To characterise the enzymes involved, a bacterial constructs was made, and the E. Coli transformed with it we called RaspberrE coli. The construct consisted of an arabinose inducible expression plasmid coding for 4-coumarate coA ligase, benzalacetone synthase, and benzalacetone reductase, and the strain of E. Coli we used had the TnaA indole synthase gene knocked out to somewhat reduce the faecal odour associated with this molecule. (growing in M9 salts with casamino acids and no tryptophan eliminated the faecal odour entirely, as the smell is due entirely to tryptophan derivatives).

The RaspberrE coli were incubated for two and a half days with 3 or 9mM p-coumaric acid and 50mM arabinose to induce expression. The cultures were then shaken with ethyl acetate which was then run on an LCMS setup (which was only possible due to the kind giving of time and expertise by Ben Pilgrim at the Cambridge Department of Chemistry).

As a control, one of the tubes incubated contained a strain of E coli resistant to chloramphenicol but with none of the raspberry ketone producing genes. There was one peak that existed on the RaspberrE coli spectra but not on the control, which when run through the mass spectrometer turned out to be benzalacetone, the precursor to raspberry ketone.

None of the E. coli which also contained the benzalacetone reductase gene produced any peaks which gave mass spectrum peaks corresponding to that of raspberry ketone. These results suggest that while the 4-coumarate coA ligase and benzalacetone synthase both worked, the benzalacetone reductase, as shown once already by the Dutch research team who first cloned the enzyme’s gene sequence (led by Jules Beekwider in Wageninen, The Netherlands), does not actually reduce benzalacetone.

A comparison of bacteria incubated with p-coumaric acid. Top: Transformed with benzalacetone generator (BBa_K1484317) Bottom: Transformed with benzalacetone generator and benzalacetone reductase. No raspberry ketone was detected in either culture.