Difference between revisions of "Part:BBa K2317007"

 
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2317007 short</partinfo>
 
<partinfo>BBa_K2317007 short</partinfo>
  
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TfdB-JLU is a monooxygenase.
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TfdB-JLU is a novel 2,4-dichlorophenol hydroxylase whose amino acid sequence exhibits less than 48% homology with other known TfdBs. Compared to wild type TfdB, TfdB-JLU has a wider substrate range and higher catalysis activity.<br>
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
===Usage and Biology===
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<partinfo>BBa_K2317007 parameters</partinfo>
 
<partinfo>BBa_K2317007 parameters</partinfo>
 
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<html>
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<head></head>
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<body>
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<h3>Experiment</h3>
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<p>Because substract and production of tfdB-JLU share close maximum absorbance, along with tfdB-JLU is a NADPH dependent enzyme, we determined enzyme activity by monitoring the decrease in absorbance at 340 nm (e340 = 6,220 M-1 cm-1) following the substrate-dependent oxidation of NADPH.</p><br/>
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<p>1. Overexpression and purification of TfdB-JLU</p>
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<p>1.1 Digestion assay<p/>
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<p>&nbsp;&nbsp;To certify succeed in constructing pET28a-tfdB-JLU, digestion assay using BamHI and XbaI was performed. The product digested by BamHI and Xbal was around 1800bp as predicted 1776bp of tfdB-JLU. </p>
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<div class="pic_box center">
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<img src="https://static.igem.org/mediawiki/2017/4/46/T--Jilin_China--application08.png" width="25%"/><br />
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Figure 1. Digestion assay of pET28a-tfdB-JLU<br />
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</div>
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<p> 1.2 Overexpression and purification of TfdB-JLU<br />
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&nbsp;&nbsp;We used optimized protocol to overexpress and purify enzyme, the purified enzyme migrated as a single band with an Mw of 63 kDa on SDS-PAGE, close to the predicted 66.9 kDa Mw of TfdB-JLU plus the 6xHis-tag. </p>
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<div class="pic_box center">
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<img src="https://static.igem.org/mediawiki/2017/6/67/T--Jilin_China--application09.jpg" width="50%"/><br/>
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<div>
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Purified TfdB-JLU was eluted in 250mM imidazole elution buffer (lane 7, lane 8)<br />
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Lane 1: crude extract <br />
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Lane 2: flow through <br />
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Lane 3: 20 mM imidazole washing buffer <br />
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Lane 4: 50 mM imidazole washing buffer <br />
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Lane 5, 6: 100 mM imidazole washing buffer<br />
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Lane 7, 8: 250 mM imidazole elution buffer<br />
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Lane 9: 500 mM imidazole buffer<br />
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</div>
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Figure 2. SDS–PAGE analysis of the expressed TfdB-JLU enzyme
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</div>
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<p> 1.3 substrate activity and preference of TfdB-JLU
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&nbsp;&nbsp;TfdB-JLU has a wide substract preference among phenolic components. The activities of chlorophenol hydroxylases were determined by monitoring the decrease in absorbance at 340 nm (e340 = 6,220 M-1 cm-1) following the substrate-dependent oxidation of NADPH (Ledger et al. 2006). Unless otherwise indicated, standard enzyme activity assays were performed by incubating the purified enzyme with 0.1 mM 2,4-DCP and 0.2 mM NADPH in 50 mM sodium phosphate buffer (pH 7.5) at 25℃ in 1ml. </p>
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<p> For determining substrate specificity, the enzyme was incubated in 50 mM sodium phosphate buffer, pH 7.5, with 0.1 mM substrate under standard conditions. Relative activity is expressed as a percentage of the maximum enzyme activity towards 2,4-DCP with FAD. </p>
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<br/>
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<p>For more information, please visit <a href="http://2017.igem.org/Team:Jilin_China/Application">2017 Jilin_China wiki.</a></p><br/>
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<h3>Reference</h3>
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<p>[1] Ledger T, Pieper DH, Gonzalez B, Chlorophenol hydroxylases encoded by plasmid pJP4 differentially contribute to chlorophenoxyacetic acid degradation. Appl Environ Microbiol 2006 72:2783–2792</p>
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</body>
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</html>

Latest revision as of 02:42, 2 November 2017

TfdB-JLU

TfdB-JLU is a novel 2,4-dichlorophenol hydroxylase whose amino acid sequence exhibits less than 48% homology with other known TfdBs. Compared to wild type TfdB, TfdB-JLU has a wider substrate range and higher catalysis activity.
Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 262
    Illegal NgoMIV site found at 925
    Illegal NgoMIV site found at 951
    Illegal NgoMIV site found at 1036
    Illegal NgoMIV site found at 1576
    Illegal NgoMIV site found at 1767
  • 1000
    COMPATIBLE WITH RFC[1000]


Experiment

Because substract and production of tfdB-JLU share close maximum absorbance, along with tfdB-JLU is a NADPH dependent enzyme, we determined enzyme activity by monitoring the decrease in absorbance at 340 nm (e340 = 6,220 M-1 cm-1) following the substrate-dependent oxidation of NADPH.


1. Overexpression and purification of TfdB-JLU

1.1 Digestion assay

  To certify succeed in constructing pET28a-tfdB-JLU, digestion assay using BamHI and XbaI was performed. The product digested by BamHI and Xbal was around 1800bp as predicted 1776bp of tfdB-JLU.


Figure 1. Digestion assay of pET28a-tfdB-JLU

1.2 Overexpression and purification of TfdB-JLU
  We used optimized protocol to overexpress and purify enzyme, the purified enzyme migrated as a single band with an Mw of 63 kDa on SDS-PAGE, close to the predicted 66.9 kDa Mw of TfdB-JLU plus the 6xHis-tag.


Purified TfdB-JLU was eluted in 250mM imidazole elution buffer (lane 7, lane 8)
Lane 1: crude extract
Lane 2: flow through
Lane 3: 20 mM imidazole washing buffer
Lane 4: 50 mM imidazole washing buffer
Lane 5, 6: 100 mM imidazole washing buffer
Lane 7, 8: 250 mM imidazole elution buffer
Lane 9: 500 mM imidazole buffer
Figure 2. SDS–PAGE analysis of the expressed TfdB-JLU enzyme

1.3 substrate activity and preference of TfdB-JLU   TfdB-JLU has a wide substract preference among phenolic components. The activities of chlorophenol hydroxylases were determined by monitoring the decrease in absorbance at 340 nm (e340 = 6,220 M-1 cm-1) following the substrate-dependent oxidation of NADPH (Ledger et al. 2006). Unless otherwise indicated, standard enzyme activity assays were performed by incubating the purified enzyme with 0.1 mM 2,4-DCP and 0.2 mM NADPH in 50 mM sodium phosphate buffer (pH 7.5) at 25℃ in 1ml.

For determining substrate specificity, the enzyme was incubated in 50 mM sodium phosphate buffer, pH 7.5, with 0.1 mM substrate under standard conditions. Relative activity is expressed as a percentage of the maximum enzyme activity towards 2,4-DCP with FAD.


For more information, please visit 2017 Jilin_China wiki.


Reference

[1] Ledger T, Pieper DH, Gonzalez B, Chlorophenol hydroxylases encoded by plasmid pJP4 differentially contribute to chlorophenoxyacetic acid degradation. Appl Environ Microbiol 2006 72:2783–2792