Difference between revisions of "Part:BBa K3871001"

Revision as of 02:18, 22 October 2021

Description

mCherry is a red fluorescent protein used as a reporter derived from the fluorophore DsRed, which was originally isolated from Discosoma sea anemones. It is commonly used due to its colour and photostability compared to other monomeric fluorophores.

This part is a variant of mCherry, the sequence of which was modified to optimize its expression in Bacillus subtilis against E. coli using the Communique tool developed by the 2021 TAU Israel team. This specific variant was optimized based on codon usage bias in highly expressed genes in B. subtilis. You may read more about the optimization model here.

For more information on this part, please consult the 2021 TAU Israel wiki results page found here. Information about experimental procedures can be found here and here.

Typical Decoding Rate (TDR) [8]:

Figure 3.7: TDR scores of E. Coli genes vs. their expression levels

This measurement is based on ribosome profiling data (Ribo-Seq), which provides a snapshot of a mid-translation ribosomal position on the mRNA molecules in a cell during certain conditions.

*This index is relatively complicated because the Ribo-Seq data is hard to produce and very noisy. Additionally, the sequences optimized according to this index underperformed in the POC experiment, thus we decided not to add this optimization to our final software.

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 <br>
 <p>For more information on this part, please consult the 2021 TAU Israel wiki results page found <a href = "https://2021.igem.org/Team:TAU_Israel/Results" style="padding-right: 0px;">here.</a> Information about experimental procedures can be found <a href = "https://2021.igem.org/Team:TAU_Israel/Notebook" style="padding-right: 0px;">here</a> and <a href = "https://2021.igem.org/Team:TAU_Israel/Proof_Of_Concept" style="padding-right: 0px;">here.</a></p>
+<h3>Typical Decoding Rate (TDR) [8]:</h3>
+<div class="container">
+<center><img src="https://2021.igem.org/wiki/images/6/60/T--TAU_Israel--fig37_model.jpeg" style="max-width: 105%;"><br><span><b>Figure 3.7:</b> TDR scores of E. Coli genes vs. their expression levels</span></center><br>
+</div><p>This measurement is based on ribosome profiling data (Ribo-Seq), which provides a snapshot of a mid-translation ribosomal position on the mRNA molecules in a cell during certain conditions.&nbsp;</p>
+<p>*This index is relatively complicated because the Ribo-Seq data is hard to produce and very noisy. Additionally, the sequences optimized according to this index underperformed in the POC experiment, thus we decided not to add this optimization to our final software.</p>
+<p id="read_more_4">Read more about TDR <b>here.</b></p>
+</div>
+<div class="row" id="TDR_more" style="display: none; border: #e8e8e8 solid;">
+<ol>
+<li>&nbsp;The Ribo-Seq reads are mapped to the CDS of the proteome</li>
+<li><span style="text-decoration: underline;">Normalization </span>- The amount of reads per gene is normalized in order to neutralize bias originated in one codon being present in more highly expressed genes. The normalization is given by:<br>
+<center><img src="https://2021.igem.org/wiki/images/b/bd/T--TAU_Israel--Equation_6.jpeg"><br><span><b>Equation (6)</b></span></center>
+<p>Where <em>NFC<sub>i</sub></em> is the normalized read count of the i-th codon, <em>RC<sub>i</sub></em> is the read count of the i-th codon, and L is the number of codons that compose the gene .&nbsp;&nbsp;</p></li>
+<li><span style="text-decoration: underline;">Merging read information</span> - The normalized number of reads mapped to each codon is collected from all mRNAs mapped, and a histogram is constructed from them.</li>
+<li><span style="text-decoration: underline;">Estimation of the distribution for each codon</span> - An exponentially modified gaussian distribution (EMG) [9] is fit to the constructed curve, and the gaussian mean is extracted as the score of that codon.</li>
+<li><span style="text-decoration: underline;">Calculating the MTDR score of the gene</span> - calculate geometric mean of the typical decoding time of each codon of the gene:<br>
+<center><img src="https://2021.igem.org/wiki/images/5/52/T--TAU_Israel--Equation_7.jpeg"><br><span><b>Equation (7)</b></span></center>
+<p>Where <em>MTDR<sub>seq</sub></em> is the mean translation time of the gene, iis the typical decoding time of the i-th codon, and <em>L</em> is the number of codons that compose the gene.&nbsp;</p></li>
+</ol>
+</div>
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