Difference between revisions of "Part:BBa K5108002"

 
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<i>P. fluorescens</i> catalase KatB ORF
  
 
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===Usage and Biology===
 
===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<div style="background-color: #f8f6f5; width: 40%; border: 2px solid #000;">
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<ol style="color: black; padding: auto -1rem; margin= 0"> <b>Contents</b>
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    <li style="color: blue;">Usage and Biology</li>
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<li style="color: blue;">Sequence and Features</li>
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<li style="color: blue;">Modeling</li>
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    <li style="color: blue;">Characterization and Measurements
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        <ol>
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            <li style="color: blue;">SDS-PAGE</li>
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            <li style="color: blue;">Growth analysis</li>
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            <li style="color: blue;">Consumption analysis of sarcosine, creatine and creatinine by NMR spectroscopy</li>
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        </ol>
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    </li>
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<li style="color: blue;">Conclusion and Perspectives</li>
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    <li style="color: blue;">References</li>
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<partinfo>BBa_K5108002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5108002 SequenceAndFeatures</partinfo>
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<h2 style="color: blue;"> <b>Usage and Biology</b></h2>
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<p>KatB is a catalase that “decomposes hydrogen peroxide into water and oxygen; it serves to protect cells from the toxic effects of hydrogen peroxide.” [1] In the context of our project, we wanted to grow <i>Pseudomonas fluorescens</i> in an oxide rich medium so we decided to overexpress this catalase by cloning it into a plasmid and transforming it into the bacteria. We hoped that it would help detoxify the medium as well as detoxify any byproducts caused by other modifications done to the bacteria.</p>
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<h2 style="color: blue;"><b>Sequence and Features</b></h2>
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<p>We decided to amplify the <i>katB</i> gene from the <i>P. fluorescens</i> genome and clone it into the pSEVA244 vector, under control of the <i>Ptrc</i> promoter which is inducible with isopropyl β-D-1-thiogalactopyranoside (IPTG) (<b>Figure 1</b>).</p>
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            <img class="d-block"
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            style="width:60%;"
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            src="https://static.igem.wiki/teams/5108/lea/pseva244-katb.png"><br><br>
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            <figcaption class="normal"><span class="titre-image"><b>Figure 1: Representation of the pSEVA244-katB plasmid.</b></span></figcaption>
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<p>To create the functional vector containing KatB, the cloning of the gene into pSEVA244 linearized was performed following In-Fusion Assembly (Takara). <b>Figure 2</b> demonstrates the successful cloning by restriction digest with EcoRI and HindIII enzymes (New England Biolabs R3101S, R3104S). The construct was confirmed by Sanger sequencing (Genewyz, <b>Figure 3</b>).
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            src="https://static.igem.wiki/teams/5108/lea/digest-katb.jpg"><br><br>
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            <figcaption class="normal"><span class="titre-image"><b>Figure 2: Restriction digest of pSEVA244-katB plasmid.</b> The plasmid was digested with EcoRI and HindIII separately or in combination. The expected (left) and experimental (right) digestion patterns are shown.</span></figcaption>
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            src="https://static.igem.wiki/teams/5108/lea/sanger-sequencing-katb.png"><br><br>
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            <figcaption class="normal"><span class="titre-image"><b>Figure 3: <i>katB</i> locus’ sequencing of pSEVA244-katB plasmid.</b> The <i>katB</i> gene was sequenced by two Sanger sequencing using two flanking primers.</span></figcaption>
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<br>
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<h2 style="color: blue;"><b>Characterization and Measurements</b></h2>
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<p>The pSEVA438-MBPeGFP plasmid, originally used in <i>P. putida</i> KT2440, was employed as positive control of <i>Pm</i> promoter's inducibility in <i>P. fluorescens</i> SBW25. This construct encodes the fusion protein MBPeGFP (Maltose-Binding Protein enhanced Green Fluorescent Protein) under the control of the <i>Pm</i> promoter. Based on the results of <i>Vogeleer P. et al. (2024)</i> [2], the pSEVA438-MBPeGFP- and pSEVA244-katB-transformed <i>P. fluorescens</i> SBW25 strains were cultured in M9 minimal medium supplemented with glucose (28 mM), with or without 0.5 mM of <i>m</i>-toluic acid inducer. After incubation, a whole-protein extraction was performed for each strain to assess the level of expression, as well as the solubility of our proteins.</p>
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<div align="center">
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        <figure class="normal mx-auto">   
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            style="width:60%;"
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            src="https://static.igem.wiki/teams/5108/lea/sds-page-katb.png"><br><br>
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            <figcaption class="normal"><span class="titre-image"><b>Figure 4: SDS-PAGE of soluble and insoluble protein fractions from cultures of <i>Pseudomonas fluorescens</i> transformed with pSEVA438-MBPeGFP or pSEVA244-katB.</b> <i>P. fluorescens</i> was cultured with and without inducer, <i>m</i>-toluic acid. Arrows show expected size of MBPeGFP and the catalase (KatB).</span></figcaption>
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K5108002 parameters</partinfo>
 
<partinfo>BBa_K5108002 parameters</partinfo>
 
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Revision as of 11:39, 27 September 2024


Catalase from Pseudomonas fluorescens SBW25

P. fluorescens catalase KatB ORF


    Contents
  1. Usage and Biology
  2. Sequence and Features
  3. Modeling
  4. Characterization and Measurements
    1. SDS-PAGE
    2. Growth analysis
    3. Consumption analysis of sarcosine, creatine and creatinine by NMR spectroscopy
  5. Conclusion and Perspectives
  6. References



Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    INCOMPATIBLE WITH RFC[23]
    Unknown
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 148
    Illegal NgoMIV site found at 322
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 723
    Illegal SapI.rc site found at 1240

Usage and Biology

KatB is a catalase that “decomposes hydrogen peroxide into water and oxygen; it serves to protect cells from the toxic effects of hydrogen peroxide.” [1] In the context of our project, we wanted to grow Pseudomonas fluorescens in an oxide rich medium so we decided to overexpress this catalase by cloning it into a plasmid and transforming it into the bacteria. We hoped that it would help detoxify the medium as well as detoxify any byproducts caused by other modifications done to the bacteria.

Sequence and Features

We decided to amplify the katB gene from the P. fluorescens genome and clone it into the pSEVA244 vector, under control of the Ptrc promoter which is inducible with isopropyl β-D-1-thiogalactopyranoside (IPTG) (Figure 1).



Figure 1: Representation of the pSEVA244-katB plasmid.

To create the functional vector containing KatB, the cloning of the gene into pSEVA244 linearized was performed following In-Fusion Assembly (Takara). Figure 2 demonstrates the successful cloning by restriction digest with EcoRI and HindIII enzymes (New England Biolabs R3101S, R3104S). The construct was confirmed by Sanger sequencing (Genewyz, Figure 3).



Figure 2: Restriction digest of pSEVA244-katB plasmid. The plasmid was digested with EcoRI and HindIII separately or in combination. The expected (left) and experimental (right) digestion patterns are shown.



Figure 3: katB locus’ sequencing of pSEVA244-katB plasmid. The katB gene was sequenced by two Sanger sequencing using two flanking primers.

Characterization and Measurements

The pSEVA438-MBPeGFP plasmid, originally used in P. putida KT2440, was employed as positive control of Pm promoter's inducibility in P. fluorescens SBW25. This construct encodes the fusion protein MBPeGFP (Maltose-Binding Protein enhanced Green Fluorescent Protein) under the control of the Pm promoter. Based on the results of Vogeleer P. et al. (2024) [2], the pSEVA438-MBPeGFP- and pSEVA244-katB-transformed P. fluorescens SBW25 strains were cultured in M9 minimal medium supplemented with glucose (28 mM), with or without 0.5 mM of m-toluic acid inducer. After incubation, a whole-protein extraction was performed for each strain to assess the level of expression, as well as the solubility of our proteins.



Figure 4: SDS-PAGE of soluble and insoluble protein fractions from cultures of Pseudomonas fluorescens transformed with pSEVA438-MBPeGFP or pSEVA244-katB. P. fluorescens was cultured with and without inducer, m-toluic acid. Arrows show expected size of MBPeGFP and the catalase (KatB).