Team:Alberta/Project/Bioinformatics

From 2009.igem.org

(Difference between revisions)
Line 109: Line 109:
<font size="2">
<font size="2">
-
<P>
+
<P> In order to produce a preliminary genome list, various databases and papers were used.  These were determined through a variety of different experimental methods and have very limited overlap. Each gene must was carefully considered and a gene list of 332 genes was produced.  Additionally, 29 genes were found to be essential for the RNA's.  For more information on the Literature Reviews used, please click on the Gene Selection tab.</P>
-
Four essential gene lists from the literature were analyzed to construct a preliminary essential gene list </P>
+
-
 
+
-
<TABLE BORDER>
+
-
<TR>
+
-
<TH>Essential Gene List from the literature</TH>
+
-
<TH>Method</TH>
+
-
<TH># of Genes considered essential</TH>
+
-
<TH>% of E.coli genes considered essential</TH>
+
-
<TH># of genes unique to that list</TH>
+
-
</TR>
+
-
<TR>
+
-
<TD>Baba et al. 2006</TD>
+
-
<TD>Single gene knockout </TD>
+
-
<TD>303</TD>
+
-
<TD> 6.4%</TD>
+
-
<TD> 36</TD>
+
-
</TR>
+
-
<TR>
+
-
<TD>Gerdes et al 2003</TD>
+
-
<TD>Transposon insertions to inactivate single genes</TD>
+
-
<TD>617</TD>
+
-
<TD>13.0%</TD>
+
-
<TD> 379 </TD>
+
-
</TR>
+
-
<TR>
+
-
<TD>Gil et al 2004</TD>
+
-
<TD>Gene conservation and literature review</TD>
+
-
<TD>203</TD>
+
-
<TD>4.3%</TD>
+
-
<TD>53</TD>
+
-
</TR>
+
-
<TR>
+
-
<TD>Profiling of E.coli Chromosome (PEC) database</TD>
+
-
<TD>Literature review</TD>
+
-
<TD>302</TD>
+
-
<TD>6.3%</TD>
+
-
<TD>126</TD>
+
-
</TR>
+
-
</TABLE BORDER>
+
-
 
+
-
 
+
-
<P>These literature lists vary greatly in size and have minimal overlap. </P>
+
-
 
+
-
<b>Venn Diagram of the Number of Essential Genes Shared Between Lists in the Literature</b>
+
-
 
+
-
<img src="https://static.igem.org/mediawiki/2009/a/a1/Uofa_Venn_of_literature.png" width="450" height="450">
+
-
 
+
-
<P>The maximum number of genes in common between any two literature lists is 205, which is between Baba et al 2006 and Gerdes et al 2003. Only 48 genes were present in all four lists.</P>
+
-
 
+
-
 
+
-
</font></div>
+
-
 
+
-
      </div></div>
+
-
<b class="b4f"></b><b class="b3f"></b><b class="b2f"></b><b class="b1f"></b>
+
     </td>
     </td>
   </tr>
   </tr>
-
<tr>
 
-
<td style="height: 400; padding-left: 10px; padding-right: 10px; padding-top: 11px;">
 
-
    <b class="b1f"></b><b class="b2f"></b><b class="b3f"></b><b class="b4f"></b>
 
-
    <div class="Presentations">
 
-
    <div style="height: 400; background:#FFFFFF; colorou line-height:100% padding: 3px 0px;">
 
-
    <h1>Constructing the Biobytes Preliminary Essential Gene List</h1>
 
-
<!-- <div align="justify" style="padding-left:20px; padding-right:20px"> -->
 
-
<div align="justify">
 
-
 
-
<font size="2">
 
-
 
-
<p> The preliminary essential gene list is based on literature sources. As described in the modeling section of this wiki, the metabolic genes from this preliminary list were used as a starting point for the computer model and were greatly altered based on the model's suggestions. Non-metabolic genes in the this preliminary list were retained in the final list, described in the "Gene Selection" tab.
 
-
 
-
<h3> Criteria for Gene Selection: </h3>
 
-
<ul>
 
-
<li>Genes must be present in more than one literature list unless there is particular reason to suspect they are essential.</li>
 
-
<li>The BioBytes metabolism is modeled after the minimal metabolism proposed by Gil et al 04, with the addition of cell wall, fatty acid, heme and ubiquitin synthesis, as Gil assumed these would not be necessary in a mycoplasma like minimal cell.</li>
 
-
<li>Additional genes required for metabolism were selected based on pathway information in the Ecocyc database. Redundancy of pathways is likely why these genes don’t appear essential in Baba, Gerdes and PEC.</li>
 
-
<li>Antitoxin genes are not essential as toxin genes would not be present.</li>
 
-
</ul>
 
-
 
-
<h3>Genes for the following processes were included:</h3>
 
-
<ul>
 
-
<li>DNA replication and cell division, but no DNA repair</li>
 
-
<li>Chaperones, but no heat shock or membrane stress response system</li>
 
-
<li>Transcription</li>
 
-
<li>Translation</li>
 
-
<li>Glycolysis</li>
 
-
<li>PMF generation via an ATP synthase consuming ATP to export protons.</li>
 
-
<li>Synthesis of acetyl-CoA from pyruvate</li>
 
-
<li>Fatty acid synthesis</li>
 
-
<li>Methylerithritol pathway (for undecaprenyl phosphate and a ubiquinone side chain)</li>
 
-
<li>Synthesis of phosphatidylethanolamine, but no other phospholipids</li>
 
-
<li>Pentose phosphate pathway (converts 6 or 3 carbon sugars to 5C sugars, such as ones needed in nucleotide biosynthesis)</li>
 
-
<li>Lipoprotein synthesis (Int and lolB are lipoproteins and essential)</li>
 
-
<li>Synthesis of nucleotides (deoxy and oxy) from nucleosides</li>
 
-
<li>Attaching lipid and biotin groups to protein</li>
 
-
<li>Transport:</li>
 
-
<ul>
 
-
<li>PTC transport system (imports and phosphorylates glucose)</li>
 
-
<li>Inorganic phosphate transport</li>
 
-
<li>Nucleoside transport</li>
 
-
<li>Sec system (exports proteins to periplasm), including SRP for cotranslational membrane insertion. secB chaperone does not appear essential. There is NO tat system, which would be used to export cofactor containing folded proteins.</li>
 
-
<li>Lipoprotein transport to outermembrane</li>
 
-
<li>Glutathione transport </li>
 
-
</ul>
 
-
<li>Cofactor synthesis: </li>
 
-
<ul>
 
-
<li>Riboflavin from GTP and ribulose-5-phosphate </li>
 
-
<li>FAD from riboflavin</li>
 
-
<li>NAD from nicotinamide</li>
 
-
<li>NADPH from NAD</li>
 
-
<li>CoA from pantothenic acid</li>
 
-
<li>Methylene tetrahydroxyfolate (mTHF) from folic acid</li>
 
-
<li>S-adenosylmethionine (SAM) from methionine</li>
 
-
<li>Thiamine diphosphate (TPP) from thiamine</li>
 
-
<li>Pyridoxal-5-phosphate (PP) from pyridoxal </li>
 
-
<li>Heme from glutamate </li>
 
-
<li>Ubiquinone </li>
 
-
</ul>
 
-
</ul>
 
-
 
-
<h3>RNAs:</h3>
 
-
<P>The rrnC operon supplies all the rRNA’s and three of the tRNAs. This operon was selected because it includes the great number of tRNA’s.  To select the other tRNA’s, all tRNA’s listed as essential in PEC were first included. One tRNA was then selected for each anticodon that differed on one of the last two bases. Differences in the first base can be accommodated by anticodon 'wobble'. At least one tRNA was included for each amino acid.  </P>
 
-
 
-
 
-
<P>The complete list of essential RNA’s can be found <a href="https://2009.igem.org/Image:Uofa_RNAs_essential.xls"> here </a>. </P>
 
-
 
-
 
-
</font></div>
 
-
 
-
      </div></div>
 
-
<b class="b4f"></b><b class="b3f"></b><b class="b2f"></b><b class="b1f"></b>
 
-
    </td>
 
-
  </tr>
 
-
 
-
 
-
<tr>
 
-
<td style="height: 400; padding-left: 10px; padding-right: 10px; padding-top: 11px;">
 
-
    <b class="b1f"></b><b class="b2f"></b><b class="b3f"></b><b class="b4f"></b>
 
-
    <div class="Survey">
 
-
    <div style="height: 400; background:#FFFFFF; line-height:100% padding: 3px 0px;">
 
-
    <h1>Statistics on BioBytes Preliminary Essential Gene List</h1>
 
-
 
-
<!-- <div align="justify" style="padding-left:20px; padding-right:20px"> -->
 
-
<div align="justify">
 
-
 
-
<font size="2">
 
-
 
-
<P> Total genes in Ecoli: 4762 </P>
 
-
<P> Total protein coding genes in BioBytes preliminary essentials list: 332 </P>
 
-
<P> Total number of RNA genes in BioBytes preliminary essentials list: 29 </P>
 
-
 
-
 
-
</font></div>
 
-
 
-
      </div></div>
 
-
<b class="b4f"></b><b class="b3f"></b><b class="b2f"></b><b class="b1f"></b>
 
-
    </td>
 
-
  </tr>
 
<tr>
<tr>
<td style="height: 400; padding-left: 10px; padding-right: 10px; padding-top: 11px;">
<td style="height: 400; padding-left: 10px; padding-right: 10px; padding-top: 11px;">
Line 280: Line 126:
<font size="2">
<font size="2">
-
<p> To verify that all genes necessary for metabolism are included in our essential gene list, a computer model was developed. This model suggested many more metabolic genes to add to our gene list. See the Gene Selection tab for the final Biobytes list of essential genes, and the Modeling section for more information on how modeling was done. </p>
+
<p> To verify that all genes necessary for metabolism are included in our essential gene list, a computer model was used.  The Model was produced by the Palson group at the University of San Diego and was used in conjunction with the Cobra Toolbox developed by the System's Biology Research Group.  The model provides a new "in silico" approach to identifying essential genes. This model suggested many more metabolic genes to add to our gene list. See the Gene Selection tab for the final Biobytes list of essential genes, and the Modeling section for more information on how modeling was done. </p>
</font></div>
</font></div>

Revision as of 01:20, 16 October 2009

University of Alberta - BioBytes










































































































Why build a minimal genome?

Genomes are extremely complex. Producing a minimal genome allows for a better understanding of the function and interaction of cellular components. This better understanding can lead to optimization of synthetic processes and provides a well characterized chassis for synthetic biology. Moreover, a simplified cell can be used to study cellular processes in a controlled, characterized genetic background. Through the use of bioinformatics our team has attempted to produce a new essential gene list for a minimal genome in the ''E. coli'' bacterium.

Why We Need Bioinformatics

''E. coli'' has over 4,500 genes. The size and complexity of this genome makes it almost impossible to manually process. An ''in silico'' approach allows for this complex data to be more easily collected, manipulated, and interpreted. Bioinformatics has aided us in accomplishing the following:
  • Review lists of essential genes in the literature and existing databases and compile a preliminary essential gene list
  • Model the metabolic reactions and net growth rate of ''E.coli'' with given gene sets. This identified additional metabolic genes essential to a minimal genome.
  • Identify knock out combinations that could be tested in the wet lab, to verify the accuracy of our metabolic model.
  • Select standardized promoters and terminators that would replace the natural promoters and terminators of essential genes.
  • Determine which promoter should be used with which gene, by analyzing expression level data.
  • Design primers to amplify all essential genes from genomic DNA.
These steps have all been completed, and are described on the following pages.

Literature Review

In order to produce a preliminary genome list, various databases and papers were used. These were determined through a variety of different experimental methods and have very limited overlap. Each gene must was carefully considered and a gene list of 332 genes was produced. Additionally, 29 genes were found to be essential for the RNA's. For more information on the Literature Reviews used, please click on the Gene Selection tab.

Metabolic Modeling

To verify that all genes necessary for metabolism are included in our essential gene list, a computer model was used. The Model was produced by the Palson group at the University of San Diego and was used in conjunction with the Cobra Toolbox developed by the System's Biology Research Group. The model provides a new "in silico" approach to identifying essential genes. This model suggested many more metabolic genes to add to our gene list. See the Gene Selection tab for the final Biobytes list of essential genes, and the Modeling section for more information on how modeling was done.

Retrieved from "http://2009.igem.org/Team:Alberta/Project/Bioinformatics"