Difference between revisions of "Part:BBa K5108002"

 
 
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<partinfo>BBa_K5108002 short</partinfo>
 
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Catalse from <i>P. fluorescens</i> SBW25
  
 
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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;">Characterization and Measurements
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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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</ol>
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<partinfo>BBa_K5108002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5108002 SequenceAndFeatures</partinfo>
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<br>
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<h2 style="color: blue;"> <b>Usage and Biology</b></h2>
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<p>KatB is a catalase (<a href="https://www.uniprot.org/uniprotkb/C3K2E2/entry" target="blank">EC 1.11.1.6</a>[1]) that decomposes hydrogen peroxide into water and oxygen. In the context of our project, we wanted to grow <i>Pseudomonas fluorescens</i> in an oxide-rich medium. Therefore, we decided to overexpress this catalase to improve stress resistance by cloning its gene into a plasmid for expression in transformed bacteria. We hoped that it would help detoxify the medium as well as detoxify any byproducts caused by other modifications added to the bacteria.</p>
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<br>
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<h2 style="color: blue;"><b>Sequence and Features</b></h2>
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<p>We amplified the <i>katB</i> gene from the bacterial genome and cloned it into the pSEVA 244 vector, under control of the <i>Ptrc</i> promoter (<a href="https://parts.igem.org/Part:BBa_K3332038" target="blank">BBa_K3332038</a>) which is inducible with IPTG (<b>Figure 1</b>).</p>
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<div align="center">
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        <figure class="normal mx-auto">   
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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: Design of the expression plasmid harboring the <i>katB</i> gene</b></span></figcaption>
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        </figure>
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</div>
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<br>
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<p>To create the expression plasmid containing <i>katB</i>, we cloned the gene into the linearized pSEVA244 backbone by In-Fusion Assembly (Takara, France). <b>Figure 2</b> demonstrates the successful cloning by restriction digest with EcoRI and HindIII enzymes (New England Biolabs, France, R3101S, R3104S).
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<div align="center">
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        <figure class="normal mx-auto">   
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            <img class="d-block"
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            style="width:90%;"
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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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        </figure>
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</div>
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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 heterologous protein expression 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 inducible by <i>m</i>-toluic acid. 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 or IPTG (1 mM). 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.<br><br>
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The obtained SDS-PAGE is presented in <b>Figure 3</b>. Both soluble and insoluble fractions contain MBPeGFP, with the majority of protein being in the soluble fraction independently of the presence of the inducer. Although transcriptional leakage was clearly observed without the inducer, MBPeGFP was overproduced when the <i>Pm</i> promoter was activated with 0.5 mM of <i>m</i>-toluic acid, confirming the possibility of heterologous protein expression in <i>P. fluorescens</i> SBW25. The presence of insoluble MBPeGFP can be caused by its overexpression leading to protein aggregation.<br><br>
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SDS-PAGE analysis of the cell lysate derived from <i>P. fluorescens</i> transformed with pSEVA244 revealed a visible band at the expected size of KatB only in insoluble fractions when its expression is induced.</p>
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<div align="center">
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        <figure class="normal mx-auto">   
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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/sds-page-katb.png"><br><br>
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            <figcaption class="normal"><span class="titre-image"><b>Figure 3: 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 indicate the expected size of catalase KatB.</span></figcaption>
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        </figure>
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</div>
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<br>
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<h2 style="color: blue;"><b>Conclusion and Perspectives</b></h2>
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<p>The presence of proteins in the insoluble fraction suggests that the ​​conditions of production (e.g., inducer concentration and temperature) are not optimal for overexpressing KatB in the soluble form. After aligning the sequence of KatB with database PFAM, we noticed the presence of a peptide signal, suggesting the targeting of KatB in the periplasm. In our lysis protocol, we could not differentiate periplasmic proteins from cytoplasmic ones. To solve this problem, we could change the KatB production conditions and our lysis protocol to purify exclusively periplasmic proteins.</p>
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<br>
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<h2 style="color: blue;"><b>References</b></h2>
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<ol>
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    <i>
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<li>UniProt. (s. d.-d). https://www.uniprot.org/uniprotkb/C3K2E2/entry</i>
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<li>Vogeleer P, Millard P, Arbulú A-SO, Pflüger-Grau K, Kremling A & Létisse F (2024) Metabolic impact of heterologous protein production in Pseudomonas putida: Insights into carbon and energy flux control. Metabolic Engineering 81: 26–37</li>
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</i>
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</ol>
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</html>
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Latest revision as of 16:03, 29 September 2024


Catalase from Pseudomonas fluorescens SBW25

Catalse from P. fluorescens SBW25


    Contents
  1. Usage and Biology
  2. Sequence and Features
  3. Characterization and Measurements
  4. Conclusion and Perspectives
  5. References



Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 917
  • 23
    COMPATIBLE WITH RFC[23]
  • 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 (EC 1.11.1.6[1]) that decomposes hydrogen peroxide into water and oxygen. In the context of our project, we wanted to grow Pseudomonas fluorescens in an oxide-rich medium. Therefore, we decided to overexpress this catalase to improve stress resistance by cloning its gene into a plasmid for expression in transformed bacteria. We hoped that it would help detoxify the medium as well as detoxify any byproducts caused by other modifications added to the bacteria.


Sequence and Features

We amplified the katB gene from the bacterial genome and cloned it into the pSEVA 244 vector, under control of the Ptrc promoter (BBa_K3332038) which is inducible with IPTG (Figure 1).



Figure 1: Design of the expression plasmid harboring the katB gene

To create the expression plasmid containing katB, we cloned the gene into the linearized pSEVA244 backbone by In-Fusion Assembly (Takara, France). Figure 2 demonstrates the successful cloning by restriction digest with EcoRI and HindIII enzymes (New England Biolabs, France, R3101S, R3104S).



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.

Characterization and Measurements

The pSEVA438-MBPeGFP plasmid, originally used in P. putida KT2440, was employed as positive control of heterologous protein expression 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 inducible by m-toluic acid. 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 or IPTG (1 mM). 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.

The obtained SDS-PAGE is presented in Figure 3. Both soluble and insoluble fractions contain MBPeGFP, with the majority of protein being in the soluble fraction independently of the presence of the inducer. Although transcriptional leakage was clearly observed without the inducer, MBPeGFP was overproduced when the Pm promoter was activated with 0.5 mM of m-toluic acid, confirming the possibility of heterologous protein expression in P. fluorescens SBW25. The presence of insoluble MBPeGFP can be caused by its overexpression leading to protein aggregation.

SDS-PAGE analysis of the cell lysate derived from P. fluorescens transformed with pSEVA244 revealed a visible band at the expected size of KatB only in insoluble fractions when its expression is induced.



Figure 3: 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 indicate the expected size of catalase KatB.

Conclusion and Perspectives

The presence of proteins in the insoluble fraction suggests that the ​​conditions of production (e.g., inducer concentration and temperature) are not optimal for overexpressing KatB in the soluble form. After aligning the sequence of KatB with database PFAM, we noticed the presence of a peptide signal, suggesting the targeting of KatB in the periplasm. In our lysis protocol, we could not differentiate periplasmic proteins from cytoplasmic ones. To solve this problem, we could change the KatB production conditions and our lysis protocol to purify exclusively periplasmic proteins.


References

  1. UniProt. (s. d.-d). https://www.uniprot.org/uniprotkb/C3K2E2/entry
  2. Vogeleer P, Millard P, Arbulú A-SO, Pflüger-Grau K, Kremling A & Létisse F (2024) Metabolic impact of heterologous protein production in Pseudomonas putida: Insights into carbon and energy flux control. Metabolic Engineering 81: 26–37