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From 2014.igem.org

Results

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.

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.

Promoter identification

To identify potential promoter regions in Marchantia's genome, we looked for gene sequences from other species, mainly Arabidopsis thaliana and Physcomitrella patens. We were particularly interested in genes that were:

• Nitrate inducible
• Sulphate inducible
• Phosphate inducible
• Circadian rythm driven
• Metabolism related
• Development related
• Light responsive
• Development
• Standard templates for proteins
• Standard templates for genes

Reading recent research papers in search for these genes and looking for their sequences on GenBank and ThaleMine, we constructed a list of protein sequences to compare to the Marchantia predicted scaffolds. (For genes where only the nucleotide sequence was available, instead of the protein sequence, a C++ code was written to perform the translation). We ran a tblastn search in BLAST for the best matches between our candidate proteins and the Marchantia scaffolds. Then, for the best matches (~60% and above, with some judging by eye), we annotated the 2kb upstream of the start codon as a potential promoter region.

Example of a BLAST hit, matching an inducible nitrate transporter sequence to a Marchantia gene

We identified 30 candidate promoters this way, that we are planning to screen by inserting in a construct driving the yellow fluorescent protein Venus. For each promoter, we will make a construct with and one without amplification by GAL4 and GAL4 UAS, to evaluate the promoter strength and get around any leakages due to the use of GAL4.