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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)