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Team:Cambridge-JIC/Marchantia/Promoter
From 2014.igem.org
One of the key aims for our project is to introduce Marchantia polymorpha to iGEM with a toolset that enables future teams to develop it further and capitalise on its benefits. So given the limited knowledge about its genetic makeup at present, we have sought to find possible inducible promoters in Marchantia that could be used for parts.
Method
Our dataset was made of large gap read mapping transcripts obtained by mRNA sequencing conducted by the Haseloff Lab on the m. polymorpha Cam strain. Open Reading Frame (ORF) and Coding Sequence (CDS) predictions were made using the CLC bio Transcript Discovery plugin[1].
We used ran blastx on our data [2] to verify the validity of the predicted ORFs and to compare the sequences with the proteins present in Arabidopsis3. A list of candidate genes was compiled from the results and this list was used to calculate a frequency table of codon usage for m.polymorpha.
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.
References
1. Qiagen, CLC bio Transcript Discovery ®, http://www.clcbio.com/clc-plugin/transcript-discovery/#description back to top
2. NCBI Blast ®, http://blast.ncbi.nlm.nih.gov/Blast.cgi back to top
3. Turmel M, Otis C and Lemieux C. 2003. The Mitochondrial Genome of Chara vulgaris: Insights into the Mitochondrial DNA Architecture of the Last Common Ancestor of Green Algae and Land Plants. The Plant Cell 15(8), pp. 1888-1903. back to top
4. Cantarel BL, Morrison HG, Pearson W. 2006. Exploring the Relationship between Sequence Similarity and Accurate Phylogenetic Trees. Molecular Biology and Evolution 23(11), pp. 2090-2100. back to top
5. Wellman CH, Osterloff PL, Mohiuddin U. 2003. Fragments of the earliest land plants. Nature 425, pp. 282-285. back to top
