Difference between revisions of "Part:BBa K500000:Experience"

 
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===User Reviews===
 
===User Reviews===
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<h3><b> Cloning of Lignin Peroxidase - BBa_K500000 </b></h3>
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Lignin peroxidase is known to have dye-decolorising activity, being particularly effective when the substrates are sulphonated azo-dyes. This led us to investigate whether we could incorporate the <a href="http://2014.igem.org/Team:UCL/Project/Biobricks">Lignin Peroxidase</a>
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<a href="https://parts.igem.org/Part:BBa_K500000">(BBa_K500000)</a> BioBrick submitted by Tianjin iGEM 2010 into our project and provide further characterisation; there was no available data in the Registry regarding to the functionality or viability of this fungal part in <em>E. coli</em>.
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<br><br>The sequence for this part, found here (insert link), was codon-optimized for <em>E. coli</em> K12 and synthesis was requested along with the BioBrick prefix and suffix. While we were able to get this fragment synthesised and sent to us, the toxicity of the DNA fragment in <em>E.coli</em> prevented us from obtaining any decolourisation data. However, we were still able to further characterise the experimental issues of <a href="https://parts.igem.org/Part:BBa_K500000">(BBa_K500000)</a> in E.coli.
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<br><br>The gene synthesis was carried out by GeneOracle, who provided us with data for the toxicity issues that they encountered. In summary, in-vitro synthesis presented no particular problems, but when the sequence was cloned into the destination strain, the sequence was modified with a number of mutations, deletions, and modifications, that made it impossible to recover the original gene. Cloning was attempted on a pGOv4 plasmid; detailed information about this plasmid can be found in the files below.
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<a href="https://static.igem.org/mediawiki/2014/c/c3/UCL2014-PGOv4_Diagram.pdf">pGOv4 Diagram</a>
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<a href="https://static.igem.org/mediawiki/2014/5/58/UCL2014-PGOV4_Info.pdf">pGOv4 Information</a>
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<img src="https://static.igem.org/mediawiki/2014/b/b6/Ligper_fig1.png "width="600" height="350">
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<b>Figure 1 - Lignin Peroxidase <a href="https://parts.igem.org/Part:BBa_K500000">BBa_K500000</a> BioBrick is toxic in E.coli DH5α. </b> Graph showing that while it was possible to confirm the successful synthesis of BBa_K500000 in vitro, it was not possible to carry out any in vivo transformations of the DNA fragment into E.coli DH5α.
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<br><br>
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The toxicity issues that arise when trying to express this fungal enzyme in <em>E. coli</em> can explain why no previous attempt to characterise the part recorded in the registry have been successful. Further characterisation of this toxicity, as well as attempting to troubleshoot it, could involve the subcloning of the part into a low-expression vector.
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<br><br>
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The inability to characterise the decolourising abilities of Lignin Peroxidase made us look towards a bacterial alternative; Bacillus subtilis dye-decolorising peroxidase. This enzyme, described in literature as the bacterial equivalent of lignin peroxidase due to the similarity of their mechanisms was cloned successfully, and the data regarding its toxicity and ability to decolorise azo-dyes is developed below.
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Revision as of 18:08, 21 October 2014


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K500000

User Reviews

Cloning of Lignin Peroxidase - BBa_K500000



Lignin peroxidase is known to have dye-decolorising activity, being particularly effective when the substrates are sulphonated azo-dyes. This led us to investigate whether we could incorporate the Lignin Peroxidase (BBa_K500000) BioBrick submitted by Tianjin iGEM 2010 into our project and provide further characterisation; there was no available data in the Registry regarding to the functionality or viability of this fungal part in E. coli.

The sequence for this part, found here (insert link), was codon-optimized for E. coli K12 and synthesis was requested along with the BioBrick prefix and suffix. While we were able to get this fragment synthesised and sent to us, the toxicity of the DNA fragment in E.coli prevented us from obtaining any decolourisation data. However, we were still able to further characterise the experimental issues of (BBa_K500000) in E.coli.

The gene synthesis was carried out by GeneOracle, who provided us with data for the toxicity issues that they encountered. In summary, in-vitro synthesis presented no particular problems, but when the sequence was cloned into the destination strain, the sequence was modified with a number of mutations, deletions, and modifications, that made it impossible to recover the original gene. Cloning was attempted on a pGOv4 plasmid; detailed information about this plasmid can be found in the files below.

pGOv4 Diagram
pGOv4 Information



Figure 1 - Lignin Peroxidase BBa_K500000 BioBrick is toxic in E.coli DH5α. Graph showing that while it was possible to confirm the successful synthesis of BBa_K500000 in vitro, it was not possible to carry out any in vivo transformations of the DNA fragment into E.coli DH5α.

The toxicity issues that arise when trying to express this fungal enzyme in E. coli can explain why no previous attempt to characterise the part recorded in the registry have been successful. Further characterisation of this toxicity, as well as attempting to troubleshoot it, could involve the subcloning of the part into a low-expression vector.

The inability to characterise the decolourising abilities of Lignin Peroxidase made us look towards a bacterial alternative; Bacillus subtilis dye-decolorising peroxidase. This enzyme, described in literature as the bacterial equivalent of lignin peroxidase due to the similarity of their mechanisms was cloned successfully, and the data regarding its toxicity and ability to decolorise azo-dyes is developed below.

UNIQc5588475f1c76376-partinfo-00000001-QINU UNIQc5588475f1c76376-partinfo-00000002-QINU