Difference between revisions of "Team:Gifu/More/"

 
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<div id="main">
 
<div id="main">
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<blockquote>
 
<blockquote>
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<center><font size="20" face="Century">InterLab Study</font></center><br><br>
  
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<h1 id="theme"> <center>Project</center> </h1>
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<font size="6" face="Century">Equipment</font>
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<h3 class="theme2">Circular mRNA -the world&acute;s longest protein-</h3>
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<hr width="100%" ><br>
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<ul>
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<li> <font size="5" face="Arimo"> BioSHAKER TA-12R<br></font> </li>
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<font size="4" face="Century">
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use for shaking culture</font>
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<br><br><br><br>
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<li> <font size="5" face="Century"> Safire ART-NR:F129013<br></font> </li>
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<font size="4" face="Century">
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use for measurement of absorbance</font>
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<br><br><br><br><br>
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</ul>
  
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<h4>Project Description</h4>
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<font size="6" face="Century">Method of Measurement</font>
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<p>&nbsp;&nbsp; This year, the theme of iGEM Gifu is to synthesize a long chain protein with circular mRNA. It is a sequel to our theme of the last year.</p><br>
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<hr width="100%" ><br>
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&nbsp;&nbsp; Here, we explain our theme of the last year.
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<ul>
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The circular mRNA is expressed in <i>E. coli</i> by using a mechanism of self-splicing.
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<font size="4" face="Century">
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In <i>td</i> gene of T4 phage, there is the group 1 intron. A part of the RNA transcribed from the group 1 intron is a ribozyme which catalyzes a splicing reaction, and this RNA splices itself.
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<p>&nbsp;&nbsp;We constructed 5 plasmids [positive control, negative control, device1~3] by using BioBricks and then transformed to <i>E. coli</i> K-12 JM109. We checked them by colony direct PCR and plasmids sequence. We cultivated correct bacterias by shaking until OD660:0.5. Cultivated mediums were　transferred to 96-well plate by 100mL. We read the absorbance.</p>
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The circular mRNA can be expressed in <i>E. coli</i> by cloning a domain which functions as ribozyme for splicing of the <i>td</i> gene, and putting it in the plasmid. This method is called “PIE method.”
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<br>
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The circular mRNA without the termination codon sequence can be used to synthesize protein semi-permanently.<br><br>
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&nbsp;&nbsp;Setting of plate reader is following:<br>
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<p>&nbsp;&nbsp; We succeeded in expression of the circular mRNA, and synthesizing long chain protein last year. But the long chain protein synthesized in last year was not functional because its holding had lost shape. So, this year, we design a linker sequence and synthesize a functional long chain protein. On the other hand, the probability was just a few percent that the cyclization happens. It is thought that the ribozyme site of the splicing is far from the reactive site, and it lowers the probability. So we’ll design complemental sequence near the ribozyme site and the reactive site of splicing to make them accessible to each other, and raise the probability of the cyclization in the theme.</p>
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<img src="https://static.igem.org/mediawiki/2015/2/20/Set_safari_gify.png" border="0" width="395" height="220">
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<br><br><br><br>
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<b>Sequence data</b><br>
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<br>
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・device 1 : J23101+ I13504<br>
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<img src="https://static.igem.org/mediawiki/2015/6/6b/Sequence_data1.png" width="800"><br><br>
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<br>
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・device 2 : J23106+ I13504<br>
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<img src="https://static.igem.org/mediawiki/2015/6/60/Sequence_data2.png" width="800"><br><br>
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<br>
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・device 3 : J23117+ I13504<br>
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<img src="https://static.igem.org/mediawiki/2015/c/c7/Sequence_data3.png" width="800"><br><br>
  
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<br>These sequence datas are correct.<br></font>
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</ul>
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<br><br>
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<font size="6" face="Century">Result</font>
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<hr width="100%" ><br>
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<ul>
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<li> <font size="5" face="Century"> Definition<br></font> </li>
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<font size="4" face="Century">
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<p>&nbsp;&nbsp;We expressed absorbance as relative value for　positive control. <br>This expression is following:<br></p></font>
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<img src="https://static.igem.org/mediawiki/2015/8/85/Siki-gifu.png" border="0" width="211" height="57"><br>
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&nbsp;&nbsp;Sample : Absorbance in sample<br>
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&nbsp;&nbsp;NCav : Average of absorbance in negative control<br>
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&nbsp;&nbsp;PCav : Average of absorbance in positive control<br>
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<br>
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<br>
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<img src="https://static.igem.org/mediawiki/2015/a/a7/Interlab_data.jpg" width="830"><br><br><br><br>
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<li> <font size="5" face="Century"> Value<br></font> </li>
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<font size="4" face="Century">
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<p>Relative absorbance values for positive control are shown below. <br></p>
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<br>
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<br><img src="https://static.igem.org/mediawiki/2015/d/de/Interlab_relative.jpg" width="350"></img><br>
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<br>
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<br>
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<img src="https://static.igem.org/mediawiki/2015/e/e4/GURAHU-gifu.png" border="0" width="572" height="364"> <br><br>
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                      FIG1 Relative absorbance values <br></font>
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              20K : Device1 (J23101 + I13504)<br>
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              22A : Device2 (J23106 + I13504)<br>
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              22K : Device3 (J23117 + I13504)<br><br><br>
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 +
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</ul>
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<br><br>
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<font size="6" face="Century">Discussion</font>
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<hr width="100%" ><br>
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<ul>
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<font size="4" face="Century">
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<p>&nbsp;&nbsp;The value of device1 is much higher than that of device2 and 3. This is why each device is different from promoter sequence, especially -10 and -35 region.<br><br></p>
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<img src="https://static.igem.org/mediawiki/2015/b/b4/Konnsennsasu2.png" border="0" width="413" height="105"> <br><br>
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<p>Promoter sequence in device1 resembles consensus sequence. On the other hand, device2 and 3 is not similar than device1. This is how each GFP expression is different.</p>
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<br><br><br>
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</font>
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</ul>
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<br><br>
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<br><br></font>
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<br><br>
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<br><br>
 
</blockquote>
 
</blockquote>
  
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<div class="box1"><br> &nbsp; MENU<br>
 
<div class="box1"><br> &nbsp; MENU<br>
 
<ol id="index">
 
<ol id="index">
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<li><a href="#summary">project Description &nbsp; </a></li>
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<li><a href="https://2015.igem.org/Team:Gifu/InterLab" style="text-decoration:none;" style="text-decoration:none;" >InterLab Study&nbsp;&nbsp;&nbsp;</a></li>
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<li><a href="#intro">Introduction &nbsp; </a></li>
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<li><a href="https://2015.igem.org/Team:Gifu/Safety"style="text-decoration:none;" > SAFETY&nbsp; </a></li>
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<li><a href="#flow">Project Flow &nbsp; </a></li>
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<li><a href="https://2015.igem.org/Team:Gifu/human-practice" style="text-decoration:none;" >Policy & Practice &nbsp; </a></li>
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<li><a href="#T&M">Theory and Methods &nbsp; </a></li>
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<li><a href="#experiments">Experiments &nbsp; </a></li>
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<br>
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<li><a href="#results">Results&amp;Data analysis &nbsp; </a></li>
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−
<li><a href="#futurework">Future Works &nbsp; </a></li>
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−
<li><a href="#conclusions">Conclusions &nbsp; </a></li>
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−
<li><a href="#references">References &nbsp; </a></li><br>
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</ol>
 
</ol>
  

Latest revision as of 17:26, 18 September 2015


https://static.igem.org/mediawiki/2015/d/d8/Logo_HOME.png https://static.igem.org/mediawiki/2015/a/a4/Logo_PROJECT.png https://static.igem.org/mediawiki/2015/archive/b/b9/20150720104417%21Logo_TEAM.png https://static.igem.org/mediawiki/2015/8/86/Logo_NOTE.png https://static.igem.org/mediawiki/2015/8/87/Logo_RESULT.png https://static.igem.org/mediawiki/2015/2/28/Logo_MORE.png


InterLab Study


Equipment

  • BioSHAKER TA-12R
    • use for shaking culture



  • Safire ART-NR:F129013
    • use for measurement of absorbance




Method of Measurement

      We constructed 5 plasmids [positive control, negative control, device1~3] by using BioBricks and then transformed to E. coli K-12 JM109. We checked them by colony direct PCR and plasmids sequence. We cultivated correct bacterias by shaking until OD660:0.5. Cultivated mediums were　transferred to 96-well plate by 100mL. We read the absorbance.


      Setting of plate reader is following:




    Sequence data

    ・device 1 : J23101+ I13504



    ・device 2 : J23106+ I13504



    ・device 3 : J23117+ I13504



    These sequence datas are correct.


Result

  • Definition

    •   We expressed absorbance as relative value for　positive control.
    This expression is following:
      Sample : Absorbance in sample
      NCav : Average of absorbance in negative control
      PCav : Average of absorbance in positive control






  • Value

    • Relative absorbance values for positive control are shown below.








    FIG1 Relative absorbance values
    20K : Device1 (J23101 + I13504)
    22A : Device2 (J23106 + I13504)
    22K : Device3 (J23117 + I13504)




Discussion

      The value of device1 is much higher than that of device2 and 3. This is why each device is different from promoter sequence, especially -10 and -35 region.



    Promoter sequence in device1 resembles consensus sequence. On the other hand, device2 and 3 is not similar than device1. This is how each GFP expression is different.









  MENU
  1. InterLab Study   
  2. SAFETY 
  3. Policy & Practice