Team:Cambridge-JIC/Marchantia/Promoter

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<h2>Informatics</h2>
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                        <h1>Promoter hunt</h1>
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                        <font color="black" style="BACKGROUND-COLOR: #E6E6E6">Finding new parts for <Em>Marchantia Polymorpha</Em>
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To introduce and facilitate future use of the novel chassis <Em>Marchantia polymorpha</Em>, we computationally analysed its genome to:
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<li>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;</li>
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<li>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. </li>
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<h2 id="Promoter">Promoter identification</h2>
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<p>To identify potential promoter regions in Marchantia's genome, we looked for gene sequences from other species, mainly <i>Arabidopsis thaliana</i> and <i>Physcomitrella patens</i>. We were particularly interested in genes that were:
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One of the key aims for our project is to introduce <em>Marchantia polymorpha</em> 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 <em>Marchantia</em> that could be used for parts.
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<li>Nitrate inducible</li>
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<li>Sulphate inducible</li>
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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<a href="#Footnote1">[1]</a>.
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<p>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.</p>
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We used ran blastx on our data <a href="#Footnote2">[2]</a> 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 <i>m.polymorpha</i>.
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<img src="https://static.igem.org/mediawiki/2014/5/5d/Cambridge_JIC_Blast_example.png">
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<p>Example of a BLAST hit, matching an inducible nitrate transporter sequence to a Marchantia gene</p>
<p>Example of a BLAST hit, matching an inducible nitrate transporter sequence to a Marchantia gene</p>
<p>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. </p>
<p>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. </p>
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<h4>References</h4>
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<p id="Footnote1">1. Qiagen, CLC bio Transcript Discovery ®, <a href="http://www.clcbio.com/clc-plugin/transcript-discovery/#description">http://www.clcbio.com/clc-plugin/transcript-discovery/#description</a> <a href="#">back to top</a></p>
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<p id="Footnote2">2. NCBI Blast ®, http://blast.ncbi.nlm.nih.gov/Blast.cgi <a href="#">back to top</a></p>
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<p id="Footnote3">3. Turmel M, Otis C and Lemieux C. 2003. <i>The Mitochondrial Genome of Chara vulgaris: Insights into the Mitochondrial DNA Architecture of the Last Common Ancestor of Green Algae and Land Plants.</i> The Plant Cell 15(8), pp. 1888-1903. <a href="#">back to top</a></p>
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<p id="Footnote4">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. <a href="#">back to top</a></p>
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<p id="Footnote5">5. Wellman CH, Osterloff PL, Mohiuddin U. 2003. <i>Fragments of the earliest land plants.</i> Nature 425, pp. 282-285. <a href="#">back to top</a></p>
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Revision as of 23:13, 17 October 2014

Cambridge iGEM 2014

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Promoter hunt

Finding new parts for Marchantia Polymorpha

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

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