Team:Cambridge-JIC/Results

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Our start point was the Marchantia genome and the mRNA transcriptome predicted with Geneious software (<a href = "http://www.geneious.com/">http://www.geneious.com/</a>). The data was given to us from Jim's lab by Bernardo. 99 000 ORFs were predicted, which seems too large to be a realistic number of genes expressed by this small liverwort. Half of these were only 100 amino acids long. We set the threshold for candidate genes among these at 300 amino acids, obtaining the expected normal distribution of lengths.
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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.
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Using BLAST software (<a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a>), we compared the proteins coded by these candidate genes to the proteins present in <Em>Arabidopsis thaliana</Em>, given on Araport (<a href="www.araport.org">www.araport.org</a>). Some of the sequences showed incomplete matches, indicated that our predicted ORFs should be regarded with some vigilance. The longest sequence showed a 40% match, a small number as expected.
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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.
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The relevant DNA sequences from the candidate mRNAs using the BLAST output. Then the different codons were counted in these genes, in order to obtain a codon table for Marchantia. While the table is not strikingly similar to Arabidopsis', we can note a similarity in the slight preference for C over other bases at the end of codons and that for G-p-C sites.  
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The transformation procedure yielded a large number of transformants, which were confirmed via PCR using homogenised plants as a template and chromoprotein-specific primers for each tDNA added.
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<img src="https://static.igem.org/mediawiki/2014/9/9a/Codon_table.png" width = 450> </img>
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<img src="https://static.igem.org/mediawiki/2014/5/5f/Cambridge-JIC_transformant_PCR_right_way_up.jpg" width = 450> </img>
<p>A .xls version of this table with relevant filters can be found <a href="https://static.igem.org/mediawiki/2014/4/42/Marchantia_codon_table_analysis.xls">here</a>.</p>
<p>A .xls version of this table with relevant filters can be found <a href="https://static.igem.org/mediawiki/2014/4/42/Marchantia_codon_table_analysis.xls">here</a>.</p>

Revision as of 09:28, 30 September 2014

Cambridge iGEM 2014

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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.

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

A .xls version of this table with relevant filters can be found here.

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.

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