Difference between revisions of "Part:BBa K5108002"
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<h2 style="color: blue;"> <b>Usage and Biology</b></h2>
 
<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>) 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 its native 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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<p>KatB is a catalase (<a href="https://www.uniprot.org/uniprotkb/C3K2E2/entry" target="blank">EC 1.11.1.6</a>) that decomposes hydrogen peroxide into water and oxygen. This enzyme serves to protect cells from the toxic effects of hydrogen peroxide. In the context of our project, we wanted to grow <i>Pseudomonas fluorescens</i> in an oxide rich medium so we decided to overexpress its native 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>
 
<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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<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 (<a href="https://parts.igem.org/Part:BBa_K3332038" target="blank">BBa_K3332038</a>) which is inducible with isopropyl β-D-1-thiogalactopyranoside (IPTG) (<b>Figure 1</b>).</p>
  
 
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     <i>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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<li>Washington IM & Van Hoosier G (2012) Chapter 3 - Clinical Biochemistry and Hematology. In The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents, Suckow MA Stevens KA & Wilson RP (eds) pp 57–116. Boston: Academic Press
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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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Revision as of 12:26, 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]
    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) that decomposes hydrogen peroxide into water and oxygen. This enzyme serves to protect cells from the toxic effects of hydrogen peroxide. In the context of our project, we wanted to grow Pseudomonas fluorescens in an oxide rich medium so we decided to overexpress its native 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 (BBa_K3332038) 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 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. 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 4. 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. The presence of insoluble MBPeGFP can be caused by its overexpression leading to protein aggregation.
COMPLETER AVEC RESULTATS KATB



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).

Conclusion and Perspectives

After looking at the 3D representation of this protein, we realized that it has a peptide signal, meaning that the protein is being secreted outside of the cell. If the protein is being excreted outside of the cell, it could aid plants survive on regolith by deoxidizing the medium. In order to prove that the engineered bacterium can better survive certain conditions, the oxidative impact test should be performed again as well as a viability assay on regolith. Plant growth should also be tested in the presence of P.fluorescens pSEVA244-KatB. Hopefully, plants will live longer and in better conditions with the overexpression of the catalase.

References

    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