Difference between revisions of "Team:USP-EEL-Brazil/Design"

 
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<h4 class="grey-text heading-weight" align = "left" style="font-size:40px; font-family:Broadway;">Design</h4>
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<h4 class="grey-text heading-weight" align = "left" style="font-size:40px; font-family:Broadway;">Step by Step</h4>
  
 
<p style=" margin-bottom: 50px; text-align: justify;    text-indent: 5%; ">
 
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     • <a href="https://www.ncbi.nlm.nih.gov/nuccore/166812030">Laccase from <i>Phoma sp.</i> UHH 5-1-03 (DSM 2245):</a>3073 pb - 607 aa </p>
 
     • <a href="https://www.ncbi.nlm.nih.gov/nuccore/166812030">Laccase from <i>Phoma sp.</i> UHH 5-1-03 (DSM 2245):</a>3073 pb - 607 aa </p>
 
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</br>
 
<p style=" margin-bottom: 50px; text-align: justify;    text-indent: 5%; ">To analyze and remove the introns, we used the <a href="http://www.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=gfind">FGENESH tool</a> from Softberry. After that, to verify if the gene sequence expressed a functional protein we used the <a href="https://web.expasy.org/translate/ ">Translate tool</a>from Expazy. There we confirmed our sequences as correct.
 
<p style=" margin-bottom: 50px; text-align: justify;    text-indent: 5%; ">To analyze and remove the introns, we used the <a href="http://www.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=gfind">FGENESH tool</a> from Softberry. After that, to verify if the gene sequence expressed a functional protein we used the <a href="https://web.expasy.org/translate/ ">Translate tool</a>from Expazy. There we confirmed our sequences as correct.
 
Once our laccases are from fungi and we plan to express them into a bacterial chassis, we had to remove the signal peptide. To do so, we used the <a href="http://www.cbs.dtu.dk/services/SignalP/">SignalP 4.1 tool</a> from DTU Bioinformatics .</p>
 
Once our laccases are from fungi and we plan to express them into a bacterial chassis, we had to remove the signal peptide. To do so, we used the <a href="http://www.cbs.dtu.dk/services/SignalP/">SignalP 4.1 tool</a> from DTU Bioinformatics .</p>
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<p><center>Laccase <i>Phoma sp.</i>(Lac PH):</center></p>
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<p></br></br></br><center>Laccase <i>Phoma sp.</i>(Lac PH):</center></p>
 
<img style="text-align:center; margin-bottom: 5%;" src="https://static.igem.org/mediawiki/2018/f/f7/T--USP-EEL-Brazil--Phoma.jpeg" width=100%>
 
<img style="text-align:center; margin-bottom: 5%;" src="https://static.igem.org/mediawiki/2018/f/f7/T--USP-EEL-Brazil--Phoma.jpeg" width=100%>
 
<p><center>Laccase <i>Pleurotus ostreatus</i> (Lac PL):</center></p>
 
<p><center>Laccase <i>Pleurotus ostreatus</i> (Lac PL):</center></p>
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<p style=" margin-bottom: 50px; text-align: justify;    text-indent: 5%; ">Our final biobrick construct constituted on the following parts:</p>
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<p style=" margin-bottom: 50px; text-align: justify;    text-indent: 5%; ">Our final biobrick construct for the laccase expression constituted on the following desing:</p>
 
<center><img style="text-align:center;margin-bottom: 5%;" src="https://static.igem.org/mediawiki/2018/4/44/T--USP-EEL-Brazil--Biobrick.jpeg" width=80%></center>
 
<center><img style="text-align:center;margin-bottom: 5%;" src="https://static.igem.org/mediawiki/2018/4/44/T--USP-EEL-Brazil--Biobrick.jpeg" width=80%></center>
  
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<p><center>LacI Promoter</center></p>
 
<p><center>LacI Promoter</center></p>
 
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<td><p>Obtained from the iGEM Registry: <a href="http://parts.igem.org/Part:BBa_R0010">BBa_R0010</a> and found on the 2018 DNA Distribution Kit </p></td>
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<td><p>Obtained from the iGEM Registry: <a href="http://parts.igem.org/Part:BBa_R0010">BBa_R0010</a> and found on the 2018 DNA Distribution Kit (kit plate 3, well 4G).</p></td>
 
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<td>
 
<td>
 
<p><center>RBS</center></p>
 
<p><center>RBS</center></p>
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<td><span ">Sequence obtained from iGEM Registry <a href="http://parts.igem.org/cgi/partsdb/part_info.cgi?part_name=BBa_B0030">BBa_B0030</a> and synthetised with the gene.&nbsp;&nbsp;</span></td>
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<td><p>Sequence obtained from iGEM Registry: <a href="http://parts.igem.org/cgi/partsdb/part_info.cgi?part_name=BBa_B0030">BBa_B0030</a> and synthesized with the gene.</p></td>
 
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<td>
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<p><center>Laccase <i> Pleurotus ostreatus</i> - Lac PL</center></p></td>
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<p><center>Laccase from <i> Pleurotus ostreatus</i> - Lac PL</center></p></td>
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<td><span >Designed and synthetised by the team</span></td>
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<td><p>Laccase gene from <i> Pleurotus ostreatus</i> NRRL0366:3471 (Lac PL), was designed and synthesized by the team.</p></td>
 
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<td>
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<p><center>Laccase <i> Phoma sp.</i> - Lac PL</center></p></td>
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<p><center>Laccase from <i> Phoma sp.</i> - Lac PL</center></p></td>
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<td><span >Designed and synthetised by the team</span></td>
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<td><p>Laccase gene from<i> Phoma sp.</i> UHH 5-1-03 (DSM 2245) (Lac PH), was designed and synthesized by the team.</p></td>
 
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<td>
 
<td>
 
<p><center>Terminator</center></p></td>
 
<p><center>Terminator</center></p></td>
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<td>Found on the <a href="http://parts.igem.org/Part:pSB1C3 ">pSBIC3 backbone</a></td>
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<td><p>Found on the <a href="http://parts.igem.org/Part:pSB1C3 ">pSBIC3 backbone.</a></p></td>
 
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<!-- TABELAA  FIM----------------------------------------------------------->
 
<!-- TABELAA  FIM----------------------------------------------------------->
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Evandro José Mulinari, PhD student and researcher at the São Carlos Institute of Physics of the University of São Paulo (IFSC / USP) provided the laccase from <i>Thielavia terrestris </i>(XP_003656170.1). The gene is inserted into the pETTrx-1a / LIC vector and transformed into E. coli Rosetta Gami 2 (DE3).
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Also, our instructor Evandro Mulinari provided for us a laccase from <a href="https://www.ncbi.nlm.nih.gov/protein/XP_003656170"><i>Thielavia terrestris </i>NRRL 8126</a> of his own design.  
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<p style=" margin-bottom: 50px; text-align: justify;    text-indent: 5%; ">The gene was designed in such a way that, when expressed, the protein contains peptides with cleavage sites for TEV followed by thioredoxin and 6 histidines at the N-terminus, to make the protein purification easier.</p>
 
<p><center>Laccase <i>Thielava terrestris </i>(Lac TT):</center></p>
 
<p><center>Laccase <i>Thielava terrestris </i>(Lac TT):</center></p>
 
<img style="text-align:center; margin-bottom: 5%;" src="https://static.igem.org/mediawiki/2018/c/cb/T--USP-EEL-Brazil--laccaseMuli2.jpg" width=100%>
 
<img style="text-align:center; margin-bottom: 5%;" src="https://static.igem.org/mediawiki/2018/c/cb/T--USP-EEL-Brazil--laccaseMuli2.jpg" width=100%>
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<p><center>pETTrx-1a / LIC vector:</center></p>
 
<p><center>pETTrx-1a / LIC vector:</center></p>
 
<img style="text-align:center; margin-bottom: 5%;" src="https://static.igem.org/mediawiki/2018/5/52/T--USP-EEL-Brazil--laccaseMuli.jpg" width=100%>
 
<img style="text-align:center; margin-bottom: 5%;" src="https://static.igem.org/mediawiki/2018/5/52/T--USP-EEL-Brazil--laccaseMuli.jpg" width=100%>
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<p style=" margin-bottom: 50px; text-align: justify;    text-indent: 5%; ">This laccase (Lac TT) was inserted into the pETTrx-1a / LIC vector, witch contains resistance to kanamycin and has the expression regulated by the Lac operon. The transformation was done into <i>E. coli </i>Rosetta Gami 2 (DE3).</p>
 
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<p style=" margin-bottom: 50px; text-align: justify;    text-indent: 5%; ">The gene was designed in such a way that, when expressed, the protein contains peptides with cleavage sites for VTE followed by thioredoxin and 6 histidines at the N-terminus, to make the purification easier of these proteins. The vector also contains genes of resistance to the antibiotic kanamycin and the expression is regulated by the Lac operon.
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{{USP-EEL-Brazil/footer}}
 
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Latest revision as of 02:50, 18 October 2018

Step by Step

The sequences of our target genes were found in the CAZy data bank, where laccases are identified as enzymes from the Auxiliary Activity Family 1 (AA1):


To analyze and remove the introns, we used the FGENESH tool from Softberry. After that, to verify if the gene sequence expressed a functional protein we used the Translate toolfrom Expazy. There we confirmed our sequences as correct. Once our laccases are from fungi and we plan to express them into a bacterial chassis, we had to remove the signal peptide. To do so, we used the SignalP 4.1 tool from DTU Bioinformatics .

Completed this process we removed any restriction sites for EcoRI, NotI, PstI and SpeI from the gene. For the synthesis, we inserted an RBS sequence and the biobrick prefix and suffix, obtaining the following gene features:




Laccase Phoma sp.(Lac PH):

Laccase Pleurotus ostreatus (Lac PL):

Our final biobrick construct for the laccase expression constituted on the following desing:

PART NAME
INFORMATION

LacI Promoter

Obtained from the iGEM Registry: BBa_R0010 and found on the 2018 DNA Distribution Kit (kit plate 3, well 4G).

RBS

Sequence obtained from iGEM Registry: BBa_B0030 and synthesized with the gene.

Laccase from Pleurotus ostreatus - Lac PL

Laccase gene from Pleurotus ostreatus NRRL0366:3471 (Lac PL), was designed and synthesized by the team.

Laccase from Phoma sp. - Lac PL

Laccase gene from Phoma sp. UHH 5-1-03 (DSM 2245) (Lac PH), was designed and synthesized by the team.

Terminator

Found on the pSBIC3 backbone.

Also, our instructor Evandro Mulinari provided for us a laccase from Thielavia terrestris NRRL 8126 of his own design.

The gene was designed in such a way that, when expressed, the protein contains peptides with cleavage sites for TEV followed by thioredoxin and 6 histidines at the N-terminus, to make the protein purification easier.

Laccase Thielava terrestris (Lac TT):

pETTrx-1a / LIC vector:

This laccase (Lac TT) was inserted into the pETTrx-1a / LIC vector, witch contains resistance to kanamycin and has the expression regulated by the Lac operon. The transformation was done into E. coli Rosetta Gami 2 (DE3).

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