Difference between revisions of "Team:Aachen/Basic Part"

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<a name="K2020042" class="anchor" style="color:#005C04 ;"><big><b>K2020042</b></big> - tRNA specific for tyrosine and UAG codon in <i>E. coli</i></a></br>
 
<a name="K2020042" class="anchor" style="color:#005C04 ;"><big><b>K2020042</b></big> - tRNA specific for tyrosine and UAG codon in <i>E. coli</i></a></br>
 +
All details about this part can be seen on its <a href="http://parts.igem.org/Part:BBa_K2020042"><u style="color:#0000EE;">registry page</u></a>.</br></br>
 
   </div>
 
   </div>
 +
 
   <div class=" content_area structure">
 
   <div class=" content_area structure">
 
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             <div class="single_header_title">
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             </div><p align="justify" style="padding-left:1.0cm; padding-right:1.0cm; font-size: 16px;">
 
             </div><p align="justify" style="padding-left:1.0cm; padding-right:1.0cm; font-size: 16px;">
 
<a name="K2020043" class="anchor" style="color:#005C04;"><big><b>K2020043</b></big> – tRNA synthetase specific for the ncAA AzF and UAG codon in <i>E. coli</i> → AzF-RS</a></br>
 
<a name="K2020043" class="anchor" style="color:#005C04;"><big><b>K2020043</b></big> – tRNA synthetase specific for the ncAA AzF and UAG codon in <i>E. coli</i> → AzF-RS</a></br>
 +
All details about this part can be seen on its <a href="http://parts.igem.org/Part:BBa_K2020043"><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020045" class="anchor" style="color:#005C04;"><big><b>K2020045</b></big> – tRNA synthetase specific for the ncAA NitroY and UAG codon in <i>E. coli</i> → NitroY-RS</a></br>
 
<a name="K2020045" class="anchor" style="color:#005C04;"><big><b>K2020045</b></big> – tRNA synthetase specific for the ncAA NitroY and UAG codon in <i>E. coli</i> → NitroY-RS</a></br>
 +
All details about this part can be seen on its <a href="http://parts.igem.org/Part:BBa_K2020045"><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020046" class="anchor" style="color:#005C04;"><big><b>K2020046</b></big> – tRNA synthetase specific for the ncAA CNF and UAG codon in <i>E. coli</i> → CNF-RS</a></br>
 
<a name="K2020046" class="anchor" style="color:#005C04;"><big><b>K2020046</b></big> – tRNA synthetase specific for the ncAA CNF and UAG codon in <i>E. coli</i> → CNF-RS</a></br>
 +
All details about this part can be seen on its <a href="http://parts.igem.org/Part:BBa_K2020046"><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020050" class="anchor" style="color:#005C04;"><big><b>K2020050</b></big> - tRNA synthetase specific for tyrosine and UAG codon in <i>E. coli</i> → Y-RS</a></br>
 
<a name="K2020050" class="anchor" style="color:#005C04;"><big><b>K2020050</b></big> - tRNA synthetase specific for tyrosine and UAG codon in <i>E. coli</i> → Y-RS</a></br>
 +
All details about this part can be seen on its <a href="http://parts.igem.org/Part:BBa_K2020050"><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020051" class="anchor" style="color:#005C04;"><big><b>K2020051</b></big> - mutated tRNA synthetase specific for tyrosine and UAG codon in <i>E. coli</i> → Y-RS with Y32G</a></br>
 
<a name="K2020051" class="anchor" style="color:#005C04;"><big><b>K2020051</b></big> - mutated tRNA synthetase specific for tyrosine and UAG codon in <i>E. coli</i> → Y-RS with Y32G</a></br>
 +
All details about this part can be seen on its <a href="http://parts.igem.org/Part:BBa_K2020051"><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020052" class="anchor" style="color:#005C04;"><big><b>K2020052</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 1</a></br>
 
<a name="K2020052" class="anchor" style="color:#005C04;"><big><b>K2020052</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 1</a></br>
 +
All details about this part can be seen on its <a href="http://parts.igem.org/Part:BBa_K2020052"><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020053" class="anchor" style="color:#005C04;"><big><b>K2020053</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 2</a></br>
 
<a name="K2020053" class="anchor" style="color:#005C04;"><big><b>K2020053</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 2</a></br>
 +
All details about this part can be seen on its <a href="http://parts.igem.org/Part:BBa_K2020053"><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020054" class="anchor" style="color:#005C04;"><big><b>K2020054</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 3</a></br>
 
<a name="K2020054" class="anchor" style="color:#005C04;"><big><b>K2020054</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 3</a></br>
 +
All details about this part can be seen on its <a href="http://parts.igem.org/Part:BBa_K2020054"><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020055" class="anchor" style="color:#005C04;"><big><b>K2020055</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 4</a></br>
 
<a name="K2020055" class="anchor" style="color:#005C04;"><big><b>K2020055</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 4</a></br>
 +
All details about this part can be seen on its <a href="http://parts.igem.org/Part:BBa_K2020055"><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020056" class="anchor" style="color:#005C04;"><big><b>K2020056</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 5</a></br>
 
<a name="K2020056" class="anchor" style="color:#005C04;"><big><b>K2020056</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 5</a></br>
 +
All details about this part can be seen on its <a href="http://parts.igem.org/Part:BBa_K2020056"><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020057" class="anchor" style="color:#005C04;"><big><b>K2020057</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 6</a></br>
 
<a name="K2020057" class="anchor" style="color:#005C04;"><big><b>K2020057</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 6</a></br>
 +
All details about this part can be seen on its <a href="http://parts.igem.org/Part:BBa_K2020057"><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020058" class="anchor" style="color:#005C04;"><big><b>K2020058</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 7</a></br>
 
<a name="K2020058" class="anchor" style="color:#005C04;"><big><b>K2020058</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 7</a></br>
 +
All details about this part can be seen on its <a href="http://parts.igem.org/Part:BBa_K2020058"><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020059" class="anchor" style="color:#005C04;"><big><b>K2020059</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 8</a></br>
 
<a name="K2020059" class="anchor" style="color:#005C04;"><big><b>K2020059</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 8</a></br>
 +
All details about this part can be seen on its <a href="http://parts.igem.org/Part:BBa_K2020059"><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020060" class="anchor" style="color:#005C04;"><big><b>K2020060</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 9</a></br>
 
<a name="K2020060" class="anchor" style="color:#005C04;"><big><b>K2020060</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 9</a></br>
 +
All details about this part can be seen on its <a href="http://parts.igem.org/Part:BBa_K2020060"><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020061" class="anchor" style="color:#005C04;"><big><b>K2020061</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 10</a></br>
 
<a name="K2020061" class="anchor" style="color:#005C04;"><big><b>K2020061</b></big> – tRNA synthetase specific for DMNB-serine and UAG codon in <i>E. coli</i> → version 10</a></br>
 +
All details about this part can be seen on its <a href="http://parts.igem.org/Part:BBa_K2020061"><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
         </div>
 
         </div>
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<a name="K2020003" class="anchor" style="color:#005C04 ;"><big><b>K2020003</b></big> – mutated expression system for subtilisin E in <i>E. coli</i> (S221Y)</a>
 
<a name="K2020003" class="anchor" style="color:#005C04 ;"><big><b>K2020003</b></big> – mutated expression system for subtilisin E in <i>E. coli</i> (S221Y)</a>
The named BioBrick <a href="#K2020002" style="color:#0000EE;">K2020002</a> above for the expression of subtilisin E in <i>E. coli</i> is the basic concept of this new part. Therefore it consists of the promoter <a href="http://parts.igem.org/Part:BBa_R0010"><u style="color:#0000EE;">BBa_R0010</u></a>, the ribosome binding site <a href="http://parts.igem.org/Part:BBa_B0034"><u style="color:#0000EE;">BBa_B0034</u></a>, the leader sequence pelB <a href="http://parts.igem.org/Part:BBa_J32015"><u style="color:#0000EE;">BBa_J32015</u></a>, the newly created BioBrick part <a href="http://parts.igem.org/Part:BBa_K2020000"><u style="color:#0000EE;">BBa_K2020000</u></a> and the terminator <a href="http://parts.igem.org/Part:BBa_B0010"><u style="color:#0000EE;">BBa_B0010</u></a> like the expression system itself. The whole expression is based on the usage in <i>E. coli</i>, so the sequence of the subtilisin E gene from a wild type <i>Bacillus subtilis</i> was optimized for <i>E. coli</i> codon usage. </br>
+
The named BioBrick <a href="#K2020002" style="color:#0000EE;">K2020002</a> above for the expression of subtilisin E in <i>E. coli</i> is the basic concept of this new part. Therefore, it consists of the promoter <a href="http://parts.igem.org/Part:BBa_R0010"><u style="color:#0000EE;">BBa_R0010</u></a>, the ribosome binding site <a href="http://parts.igem.org/Part:BBa_B0034"><u style="color:#0000EE;">BBa_B0034</u></a>, the leader sequence pelB <a href="http://parts.igem.org/Part:BBa_J32015"><u style="color:#0000EE;">BBa_J32015</u></a>, the newly created BioBrick <a href="http://parts.igem.org/Part:BBa_K2020000"><u style="color:#0000EE;">K2020000</u></a> and the terminator <a href="http://parts.igem.org/Part:BBa_B0010"><u style="color:#0000EE;">BBa_B0010</u></a> like the expression system itself. The whole expression is based on the usage in <i>E. coli</i>, so the sequence of the subtilisin E gene from a wild type <i>Bacillus subtilis</i> was optimized for <i>E. coli</i> codon usage. </br>
The sequence was partly ordered from IDT (<a href="http://parts.igem.org/Part:BBa_K2020001"><u style="color:#0000EE;">BBa_K2020001</u></a> + <a href="http://parts.igem.org/Part:BBa_B0010"><u style="color:#0000EE;">BBa_B0010</u></a>) and then cloned into <a href="http://parts.igem.org/Part:BBa_J04500"><u style="color:#0000EE;">BBa_J04500</u></a>, a protein expression backbone which already carries the LacI promoter <a href="http://parts.igem.org/Part:BBa_R0010"><u style="color:#0000EE;">BBa_R0010</u></a> and the ribosome binding site <a href="http://parts.igem.org/Part:BBa_B0034"><u style="color:#0000EE;">BBa_B0034</u></a>. Afterwards, a mutation in the active site of the enzyme was introduced by performing a site-directed mutagenesis. The codon AGC of serine<sup>221</sup> was substituted with TAC which codes for tyrosine.</br>
+
The sequence was partly ordered from IDT (<a href="http://parts.igem.org/Part:BBa_K2020001"><u style="color:#0000EE;">BBa_K2020001</u></a> + <a href="http://parts.igem.org/Part:BBa_B0010"><u style="color:#0000EE;">BBa_B0010</u></a>) and then cloned into <a href="http://parts.igem.org/Part:BBa_J04500"><u style="color:#0000EE;">BBa_J04500</u></a>, a protein expression backbone which already carries the LacI promoter <a href="http://parts.igem.org/Part:BBa_R0010"><u style="color:#0000EE;">BBa_R0010</u></a> and the ribosome binding site <a href="http://parts.igem.org/Part:BBa_B0034"><u style="color:#0000EE;">BBa_B0034</u></a>. Afterwards, a mutation in the active site of the enzyme was introduced by performing a site-directed mutagenesis. The codon AGC of serine<sup>221</sup> was substituted with TAC which codes for tyrosine, so serine was exchanged against tyrosine in the catalytic triade of the enzyme.</br>
By performing a site-directed mutagenesis, serine in the catalytic triade of the enzyme was exchanged against tyrosine. Therefore, the alkaline serine protease looses its proteolytic activity of non-specifically digesting proteins. Caused to its leader sequence pelB <a href="http://parts.igem.org/Part:BBa_J32015"><u style="color:#0000EE;">BBa_J32015</u></a>, the BioBrick secrets an inactive version of subtilisin E into the periplasm of the cell as soon as it is introduced into <i>E. coli</i>. </br></br>
+
We were not able to exactly detect the expression of the modified proteases via SDS gel, so we proceeded by executing a skim milk assay on agar plates containing IPTG and the needed antibiotics. Therefore, we streaked the cells containing the modified expression systems on these plates and incubated at 30°C for three days.</br>
 +
 
 +
<center><img src="https://static.igem.org/mediawiki/2016/1/10/T--Aachen--labbook_ecoli_skim_milk_mutated.png" style="width:600px;"/></center>
 +
 
 +
<figcaption style="text-align:center; font-size: 15px; padding-left:2cm;padding-right:2cm; "><b>Figure 1: Skim milk plates assay.</b> Cells containing the empty backbone (1) in comparison to either cells producing the native protease (2), cells with the SDM 1-modified (3) and the SDM 3-modified expression system (4) after incubation for 3 days at 30°C.</figcaption><br/>
 +
 
 +
<p align="justify" style="padding-left: 1.0cm; padding-right: 1.0cm; font-size:16px;">
 +
Neither the empty backbone nor the SDM 1 modified expression system did seem to cause a proteolytic activity. A clearance and therefore a proteolytic activity could only be observed for the native protease (as demonstrated in the section of BioBrick <a href="#K2020002" style="color:#0000EE;">K2020002</a>).
 +
By demonstrating that this modification doesn’t result in a clearance of the skim milk plates, we were now able to prove that serine is essential for the proteolytic activity of the protease and that exchanging it would inactivate the enzyme. Hence, we demonstrated that exchanging serine against a photo-labile, non-canonical amino acid will inactivate subtilisin E and therefore proved the principle of our project. </br>
 +
All details about this part can be seen on its <a href=”http://parts.igem.org/Part:BBa_K2020003”><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020004" class="anchor" style="color:#005C04 ;"><big><b>K2020004</b></big> – mutated expression system for subtilisin E in <i>E. coli</i> (S221X)</a></br>
 
<a name="K2020004" class="anchor" style="color:#005C04 ;"><big><b>K2020004</b></big> – mutated expression system for subtilisin E in <i>E. coli</i> (S221X)</a></br>
 +
All details about this part can be seen on its <a href=”http://parts.igem.org/Part:BBa_K2020004”><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020005" class="anchor" style="color:#005C04 ;"><big><b>K2020005</b></big> – mutated expression system for subtilisin E in <i>E. coli</i> (Y77W)</a></br>
 
<a name="K2020005" class="anchor" style="color:#005C04 ;"><big><b>K2020005</b></big> – mutated expression system for subtilisin E in <i>E. coli</i> (Y77W)</a></br>
 +
All details about this part can be seen on its <a href=”http://parts.igem.org/Part:BBa_K2020005”><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020006" class="anchor" style="color:#005C04 ;"><big><b>K2020006</b></big> – mutated expression system for subtilisin E in <i>E. coli</i> (Y77X)</a></br>
 
<a name="K2020006" class="anchor" style="color:#005C04 ;"><big><b>K2020006</b></big> – mutated expression system for subtilisin E in <i>E. coli</i> (Y77X)</a></br>
 +
All details about this part can be seen on its <a href=”http://parts.igem.org/Part:BBa_K2020006”><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
</div>
 
</div>
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<br/><p align="justify" style="padding-left:1.0cm; padding-right:1.0cm; font-size: 16px;">
 
<br/><p align="justify" style="padding-left:1.0cm; padding-right:1.0cm; font-size: 16px;">
 
<a name="K2020026" class="anchor" style="color:#005C04 ;"><big><b>K2020026</b></big> – leader sequence MFalpha (different version of the biobrick K792002)</a>
 
<a name="K2020026" class="anchor" style="color:#005C04 ;"><big><b>K2020026</b></big> – leader sequence MFalpha (different version of the biobrick K792002)</a>
This part is a different version of the leader sequence MFalpha (<a href=”http://parts.igem.org/Part:BBa_K792002”><u style="color:#0000EE;">BBa_K792002</u></a>) for <i>Saccharomyces cerevisiae</i>. It can be cloned directly in front of the protein one wants to be secreted. Therefore, it will be relocated into the medium and the tag will be cleaved off during this process. This alpha mating factors secretion tag is naturally occurring in the genome of <i>S. cerevisiae</i>. Futhermore, the sequence contained an illegal restriction site, which was corrected via PCR by us.
+
This part is a different version of the leader sequence MFalpha (<a href=”http://parts.igem.org/Part:BBa_K792002”><u style="color:#0000EE;">BBa_K792002</u></a>) for <i>Saccharomyces cerevisiae</i>. It can be cloned directly in front of the protein one wants to be secreted. Therefore, it will be relocated into the medium and the tag will be cleaved off during this process. This alpha mating factors secretion tag is naturally occurring in the genome of <i>S. cerevisiae</i>. Futhermore, the sequence contained an illegal restriction site, which was corrected via PCR by us.</br>
 
+
All details about this part can be seen on its <a href=”http://parts.igem.org/Part:BBa_K2020026”><u style="color:#0000EE;">registry page</u></a>.</br></br>
</br></br>
+
 
    
 
    
 
</div>
 
</div>
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<br/><p align="justify" style="padding-left:1.0cm; padding-right:1.0cm; font-size: 16px;">
 
<br/><p align="justify" style="padding-left:1.0cm; padding-right:1.0cm; font-size: 16px;">
 
<a name="K2020040" class="anchor" style="color:#005C04 ;"><big><b>K2020040</b></big> – screening plasmid for incorporation of non-canonical amino acids → pRXG</a>
 
<a name="K2020040" class="anchor" style="color:#005C04 ;"><big><b>K2020040</b></big> – screening plasmid for incorporation of non-canonical amino acids → pRXG</a>
This part is an improvement of the existing BioBrick <a href="http://parts.igem.org/Part:BBa_K1416004"><u style="color:#0000EE;">K1416004</u></a>, called pFRY by the iGEM Team Austin Texas 2014.</br></br>
+
This part is an improvement of the existing BioBrick <a href="http://parts.igem.org/Part:BBa_K1416004"><u style="color:#0000EE;">K1416004</u></a>, called pFRY by the iGEM Team Austin Texas 2014.</br>
 +
All details about this part can be seen on its <a href=”http://parts.igem.org/Part:BBa_K2020040”><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<a name="K2020041" class="anchor" style="color:#005C04 ;"><big><b>K2020041</b></big> – screening plasmid for incorporation of non-canonical amino acids → pRYG </a>
 
<a name="K2020041" class="anchor" style="color:#005C04 ;"><big><b>K2020041</b></big> – screening plasmid for incorporation of non-canonical amino acids → pRYG </a>
 
This part is an improvement of the existing BioBrick <a href="http://parts.igem.org/Part:BBa_K1416003"><u style="color:#0000EE;">K1416003</u></a>, called pFRYC by the iGEM Team Austin Texas 2014.</br>
 
This part is an improvement of the existing BioBrick <a href="http://parts.igem.org/Part:BBa_K1416003"><u style="color:#0000EE;">K1416003</u></a>, called pFRYC by the iGEM Team Austin Texas 2014.</br>
 +
All details about this part can be seen on its <a href=”http://parts.igem.org/Part:BBa_K2020041”><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
  
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<span style="color:#005C04;"><big><b>K2020042</b></big> – tRNA specific for tyrosine and UAG codon in <i>E. coli</i></span></br>
 
<span style="color:#005C04;"><big><b>K2020042</b></big> – tRNA specific for tyrosine and UAG codon in <i>E. coli</i></span></br>
Details about this part can be seen above in the section <a href="#basic" style="color:#0000EE;">“Best Basic Part”</a> and on its <a href=”http://parts.igem.org/Part:BBa_K2020042”><u style="color:#0000EE;">registry page</u></a>.</br></br>
+
Details about this part can be seen above in the section <a href="#basic" style="color:#0000EE;">“Best Basic Part”</a> and on its <a href="http://parts.igem.org/Part:BBa_K2020042"><u style="color:#0000EE;">registry page</u></a>.</br></br>
  
 
<span style="color:#005C04;"><big><b>K2020043</b></big> – tRNA synthetase specific for the ncAA AzF and UAG codon in <i>E. coli</i> → AzF-RS</span></br>
 
<span style="color:#005C04;"><big><b>K2020043</b></big> – tRNA synthetase specific for the ncAA AzF and UAG codon in <i>E. coli</i> → AzF-RS</span></br>

Revision as of 21:21, 18 October 2016

Welcome to iGEM Aachen 2016

Parts

BioBrick Description
K2020000 subtilisin E gene, optimized for E. coli codon usage
K2020001 subtilisin E gene, optimized for E. coli codon usage, with leader sequence pelB
K2020002 expression system for subtilisin E in E. coli
K2020003 mutated expression system for subtilisin E in E. coli (S221Y)
K2020004 mutated expression system for subtilisin E in E. coli (S221X)
K2020005 mutated expression system for subtilisin E in E. coli (Y77W)
K2020006 mutated expression system for subtilisin E in E. coli (Y77X)
K2020026 leader sequence MFalpha (different version of the biobrick K792002)
K2020040 screening plasmid for incorporation of non-canonical amino acids → pRXG (twin pFRY)
K2020041 screening plasmid for incorporation of non-canonical amino acids → pRYG (twin pFRYC)
K2020042 tRNA specific for tyrosine and UAG codon in E. coli
K2020043 tRNA synthetase specific for the ncAA AzF and UAG codon in E. coli → AzF-RS
K2020045 tRNA synthetase specific for the ncAA NitroY and UAG codon in E. coli → NitroY-RS
K2020046 tRNA synthetase specific for the ncAA CNF and UAG codon in E. coli → CNF-RS
K2020050 tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS
K2020051 mutated tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS with Y32G
K2020052 tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 1
K2020053 tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 2
K2020054 tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 3
K2020055 tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 4
K2020056 tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 5
K2020057 tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 6
K2020058 tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 7
K2020059 tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 8
K2020060 tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 9
K2020061 tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 10


K2020002 – expression system for subtilisin E in E. coli This expression system consists of the promoter BBa_R0011, the ribosome binding site BBa_B0034, the newly created BioBrick part BBa_K2020001 and the terminator BBa_B0010. BioBrick K2020001 is a composite part itself and includes the secretion tag pelB (BBa_J32015) and a subtilisin E gene optimized for Escherichia coli codon usage (BBa_K2020000). Once introduced into E. coli, this BioBrick is able to produce subtilisin E, an alkaline serine protease which non-specifically digest proteins, and simultaneously secret the enzyme into the periplasm of the cell. Caused by the lacI regulated promoter BBa_R0010, the expression system can be induced by addition of IPTG.
With the iGEM promoter BBa_R0011, which was integrated in our sequence at first, it was not possible to successfully express subtilisin E due to fatal mutations inside the expression system in all analyzed colonies. Either there have been single base deletions or insertions in the pro-peptide, which led to a frameshift of the whole protein, or a 23 base pair deletion in the promoter. Both types of mutations result in an incorrect expression system, so that an expression of the protease is impossible. Since the promoter BBa_R0011 is leaky and induce the expression even without addition of IPTG, it can be assumed that subtilisin E is toxic for E. coli.
Hence, we exchanged the promoter against BBa_R0010. For achieving this, we fulfilled a Polymerase Chain Reaction to extract everything but the promoter and the RBS and simultaneously extend the remaining DNA sequence with the pre-fix of iGEM. Afterwards, we assembled it with the BioBrick J04500 and in parallel cloned it into the vector pSB1C3 - by cutting RFP out of the BioBrick J04450. The implemented BioBrick J04500 itself contains another IPTG inducible promoter (BBa_R0010) and the same RBS (BBa_B0034). An expression with the newly integrated promoter BBa_R0010 led in a colony with the correct sequence in opposition to our trial of gaining a positive clone while working with the first promoter BBa_R0011.
We continued our experiments by performing a skim milk assay on agar plates. Therefore, we poured LB skim milk agar plates containing IPTG and the needed antibiotic and streaked the E. coli BL21 cells containing the plasmid with the expression system.

Figure 1: Skim milk plates assay. Cells containing the empty backbone (left) and cells containing the expression system for native subtilisin E (right) after incubation for 3 days at 30°C.

Comparing the clearance of the skim milk plates, a proteolytic activity could be proven for the cells containing the expression system for native subtilisin E. As a results, we concluded that within three days these cells are able to produce the native protease which will then digest the skim milk in the agar plates, resulting in a clearance.
In conclusion, we were able to express subtilisin E in E. coli and to prove its proteolytic activity via skim milk assay.
All details about this part can be seen on its registry page.

K2020043 – tRNA synthetase specific for the ncAA AzF and UAG codon in E. coli → AzF-RS
All details about this part can be seen on its registry page.

K2020045 – tRNA synthetase specific for the ncAA NitroY and UAG codon in E. coli → NitroY-RS
All details about this part can be seen on its registry page.

K2020046 – tRNA synthetase specific for the ncAA CNF and UAG codon in E. coli → CNF-RS
All details about this part can be seen on its registry page.

K2020050 - tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS
All details about this part can be seen on its registry page.

K2020051 - mutated tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS with Y32G
All details about this part can be seen on its registry page.

K2020052 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 1
All details about this part can be seen on its registry page.

K2020053 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 2
All details about this part can be seen on its registry page.

K2020054 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 3
All details about this part can be seen on its registry page.

K2020055 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 4
All details about this part can be seen on its registry page.

K2020056 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 5
All details about this part can be seen on its registry page.

K2020057 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 6
All details about this part can be seen on its registry page.

K2020058 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 7
All details about this part can be seen on its registry page.

K2020059 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 8
All details about this part can be seen on its registry page.

K2020060 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 9
All details about this part can be seen on its registry page.

K2020061 – tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 10
All details about this part can be seen on its registry page.

Expression in E. coli

Basic Building Blocks


K2020000 – subtilisin E gene, optimized for E. coli codon usage The gene of this BioBrick can be used to express subtilisin E, which is an alkaline serine protease, which non specifically digest proteins, in Escherichia coli. To use this part a suitable leader sequence has to be placed in front as the sequence of this BioBrick does not contain a start codon. Its sequence was originally obtained from a wild type Bacillus subtilis but was codon optimized for E. coli.
All details about this part can be seen on its registry page and in the section of the related BioBrick K2020002.

K2020001 – subtilisin E gene, optimized for E. coli codon usage, with leader sequence pelB This composite part consists of the leader sequence pelB (BBa_J32015) and the subtilisin E gene (K2020000) from our first BioBrick. It can be used to express subtilisin E in Escherichia coli and simultaneously secret the enzyme into the periplasm of the cell. Subtilisin E is an alkaline serine protease which non-specifically digests proteins.
To generate a functional coding sequence that can be expressed in E. coli the leader sequence pelB, which begins with a start codon, was placed in front of the subtilisin E gene.
Caused to subtilisin E’s partly toxicity for E. coli, this BioBrick should be cloned into an expression system with an inducible promoter. This has to hinder the organism in the expression of the protease while its growing period. With the iGEM promoter BBa_R0011 it was not possible to successfully express subtilisin E. Hence, we exchange the promoter against BBa_R0010.
All details about this part can be seen on its registry page and in the section of the related BioBrick K2020002.

K2020002 – expression system for subtilisin E in E. coli
Details about this part can be seen above in the section Best New Composite Part and on its registry page.

Mutated Versions


K2020003 – mutated expression system for subtilisin E in E. coli (S221Y) The named BioBrick K2020002 above for the expression of subtilisin E in E. coli is the basic concept of this new part. Therefore, it consists of the promoter BBa_R0010, the ribosome binding site BBa_B0034, the leader sequence pelB BBa_J32015, the newly created BioBrick K2020000 and the terminator BBa_B0010 like the expression system itself. The whole expression is based on the usage in E. coli, so the sequence of the subtilisin E gene from a wild type Bacillus subtilis was optimized for E. coli codon usage.
The sequence was partly ordered from IDT (BBa_K2020001 + BBa_B0010) and then cloned into BBa_J04500, a protein expression backbone which already carries the LacI promoter BBa_R0010 and the ribosome binding site BBa_B0034. Afterwards, a mutation in the active site of the enzyme was introduced by performing a site-directed mutagenesis. The codon AGC of serine221 was substituted with TAC which codes for tyrosine, so serine was exchanged against tyrosine in the catalytic triade of the enzyme.
We were not able to exactly detect the expression of the modified proteases via SDS gel, so we proceeded by executing a skim milk assay on agar plates containing IPTG and the needed antibiotics. Therefore, we streaked the cells containing the modified expression systems on these plates and incubated at 30°C for three days.

Figure 1: Skim milk plates assay. Cells containing the empty backbone (1) in comparison to either cells producing the native protease (2), cells with the SDM 1-modified (3) and the SDM 3-modified expression system (4) after incubation for 3 days at 30°C.

Neither the empty backbone nor the SDM 1 modified expression system did seem to cause a proteolytic activity. A clearance and therefore a proteolytic activity could only be observed for the native protease (as demonstrated in the section of BioBrick K2020002). By demonstrating that this modification doesn’t result in a clearance of the skim milk plates, we were now able to prove that serine is essential for the proteolytic activity of the protease and that exchanging it would inactivate the enzyme. Hence, we demonstrated that exchanging serine against a photo-labile, non-canonical amino acid will inactivate subtilisin E and therefore proved the principle of our project.
All details about this part can be seen on its registry page.

K2020004 – mutated expression system for subtilisin E in E. coli (S221X)
All details about this part can be seen on its registry page.

K2020005 – mutated expression system for subtilisin E in E. coli (Y77W)
All details about this part can be seen on its registry page.

K2020006 – mutated expression system for subtilisin E in E. coli (Y77X)
All details about this part can be seen on its registry page.

Expression in S. cerevisiae

Improvement of an Existing Part


K2020026 – leader sequence MFalpha (different version of the biobrick K792002) This part is a different version of the leader sequence MFalpha (BBa_K792002) for Saccharomyces cerevisiae. It can be cloned directly in front of the protein one wants to be secreted. Therefore, it will be relocated into the medium and the tag will be cleaved off during this process. This alpha mating factors secretion tag is naturally occurring in the genome of S. cerevisiae. Futhermore, the sequence contained an illegal restriction site, which was corrected via PCR by us.
All details about this part can be seen on its registry page.

Evolution of a New Synthetase

Screening System


K2020040 – screening plasmid for incorporation of non-canonical amino acids → pRXG This part is an improvement of the existing BioBrick K1416004, called pFRY by the iGEM Team Austin Texas 2014.
All details about this part can be seen on its registry page.

K2020041 – screening plasmid for incorporation of non-canonical amino acids → pRYG This part is an improvement of the existing BioBrick K1416003, called pFRYC by the iGEM Team Austin Texas 2014.
All details about this part can be seen on its registry page.

tRNA and Synthetases


K2020042 – tRNA specific for tyrosine and UAG codon in E. coli
Details about this part can be seen above in the section “Best Basic Part” and on its registry page.

K2020043 – tRNA synthetase specific for the ncAA AzF and UAG codon in E. coli → AzF-RS
Details about this part can be seen above in the section “Parts Collection” and on its registry page.

K2020045 – tRNA synthetase specific for the ncAA NitroY and UAG codon in E. coli → NitroY-RS
Details about this part can be seen above in the section “Parts Collection” and on its registry page.

K2020046 – tRNA synthetase specific for the ncAA CNF and UAG codon in E. coli → CNF-RS
Details about this part can be seen above in the section “Parts Collection” and on its registry page.

K2020050 – tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS
Details about this part can be seen above in the section “Parts Collection” and on its registry page.

K2020051 – mutated tRNA synthetase specific for tyrosine and UAG codon in E. coli → Y-RS with Y32G
Details about this part can be seen above in the section “Parts Collection” and on its registry page.

K2020052 to K2020061 - tRNA synthetase specific for DMNB-serine and UAG codon in E. coli → version 1 to 10
Details about these parts can be seen above in the section “Parts Collection” and on their registry pages.