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Team:Cambridge-JIC/Marchantia/Promoter
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Revision as of 16:00, 16 October 2014
Informatics
To introduce and facilitate future use of the novel chassis Marchantia polymorpha, we computationally analysed its genome to:- find out the most efficient codon usage in order to optimise our Marchantia specific registry parts and facilitate all future synthetic biology work on Marchantia;
- submit a small library of Marchantia promoters to the iGEM registry, in particular looking for those which are strong, inducible, tissue-specific or expressed in an early development stage.
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.
