Difference between revisions of "Part:BBa K5108000"
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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>Hfq is an RNA-binding protein. This protein interacts with mRNAs, either alone or together with regulatory small noncoding RNAs (sRNAs), affecting mRNA translation and degradation in bacteria [1]. Overexpression of this protein has been linked to better resistance in hostile environments [2]. In the context of our project, we cloned this gene into a plasmid containing another stress-resistance gene, <i>rpoS</i>, in order to promote the stress response of <i>Pseudomonas fluorescens</i>, so that it would better survive in hostile environments.</p>
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<p>Hfq is an RNA-binding protein. This protein interacts with mRNAs, either alone or together with regulatory small noncoding RNAs (sRNAs), affecting mRNA translation and degradation in bacteria [1]. Overexpression of this protein has been linked to better resistance in hostile environments [2]. In the context of our project, we cloned this gene into a plasmid containing another stress-resistance gene, <i>rpoS</i>, in order to promote the general stress response of <i>Pseudomonas fluorescens</i>, so that it would better survive in hostile environments.</p>
 
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             src="https://static.igem.wiki/teams/5108/lea/hfq-cloning.png"><br><br>  
 
             src="https://static.igem.wiki/teams/5108/lea/hfq-cloning.png"><br><br>  
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             <figcaption class="normal"><span class="titre-image"><b>Figure 1: Representation of the cloning strategy for pSEVA438-Ptet-hfq-rpoS plasmid.</b></span></figcaption>
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             <figcaption class="normal"><span class="titre-image"><b>Figure 1: Representation of the design of pSEVA438-Ptet-hfq-rpoS plasmid.</b></span></figcaption>
 
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<p>To create the functional vector containing <i>hfq</i>, cloning of the construct into a linearized pSEVA438 was performed following 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). The construct was confirmed by Sanger sequencing (Genewyz, Germany, Figure 3).</p>
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<p>To create the functional vector containing <i>hfq</i>, cloning of the construct into a linearized pSEVA438 was performed following 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).
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            src="https://static.igem.wiki/teams/5108/lea/hfq-sequencing.png"><br><br>
 
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            <figcaption class="normal"><span class="titre-image"><b>Figure 3: <i>hfq</i> locus’ sequencing of pSEVA438-Ptet-hfq-rpoS plasmid.</b> The <i>hfq</i> gene was sequenced by one Sanger sequencing using flanking primer. Sequencing analysis was done by MAFFT alignment on Bencheling.
 
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<h2 style="color: blue;">Conclusion and Perspectives</h2>
 
<h2 style="color: blue;">Conclusion and Perspectives</h2>
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<p>Unfortunately, the cloning of the <i>rpoS</i> gene in the same construct as <i>hfq</i> was unsuccessful, the sequencing revealed detrimental mutations. For this reason, and for lack of time, we were unable to characterize the effect of an overexpressed Hfq protein in <i>P. fluorescens</i> and its resistance to stressful environments.  
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<p>Unfortunately, the cloning of the <i>rpoS</i> gene in the same construct as <i>hfq</i> was unsuccessful. Sequencing data revealed deleterious mutations. For this reason, and for lack of time, we were unable to overexpress Hfq protein in <i>P. fluorescens</i> and assay the resistance it would confer against stressful environments.  
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We encourage future iGEM teams to explore the effect of this protein overexpression on the stress response, particularly osmotic stress and alkaline pH. Of interest, we previously characterized the growth of <i>P. fluorescens</i> SBW25 on M9 minimal medium supplemented with glucose with different concentrations of NaCl (<a href="https://2024.igem.wiki/toulouse-insa-ups/home" target="blank">iGEM Toulouse INSA-UPS 2024</a>).</p>
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We encourage future iGEM teams to explore the effect of this protein on the bacterial stress response, particularly against osmotic stress and alkaline pH.</p>
 
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Revision as of 15:32, 29 September 2024


RNA binding protein Hfq from Pseudomonas fluorescens ATCC13525

P. fluorescens Hfq ORF



Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

Hfq is an RNA-binding protein. This protein interacts with mRNAs, either alone or together with regulatory small noncoding RNAs (sRNAs), affecting mRNA translation and degradation in bacteria [1]. Overexpression of this protein has been linked to better resistance in hostile environments [2]. In the context of our project, we cloned this gene into a plasmid containing another stress-resistance gene, rpoS, in order to promote the general stress response of Pseudomonas fluorescens, so that it would better survive in hostile environments.


Sequence and Features

The hfq gene was synthesized in antisense with rpoS gene, both separated by a bi-directional terminator, LUZ7 T50 (BBa_K4757058). hfq gene was cloned downstream the hcnA CHAO RBS (BBa-K5108007) to allow its expression in P. fluorescens. This construct was cloned into a pSEVA438 plasmid under the control of the Pm promoter, inducible with m-toluic acid.



Figure 1: Representation of the design of pSEVA438-Ptet-hfq-rpoS plasmid.

To create the functional vector containing hfq, cloning of the construct into a linearized pSEVA438 was performed following 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 pSEVA438-Ptet-hfq-rpoS plasmid. The plasmid was digested with EcoRI and HindIII separately or in combination. The expected (left) and experimental (right) digestion patterns are shown.

Conclusion and Perspectives

Unfortunately, the cloning of the rpoS gene in the same construct as hfq was unsuccessful. Sequencing data revealed deleterious mutations. For this reason, and for lack of time, we were unable to overexpress Hfq protein in P. fluorescens and assay the resistance it would confer against stressful environments. We encourage future iGEM teams to explore the effect of this protein on the bacterial stress response, particularly against osmotic stress and alkaline pH.


References

  1. Trouillon J, Han K, Attrée I & Lory S (2022) The core and accessory Hfq interactomes across Pseudomonas aeruginosa lineages. Nat Commun 13: 1258
  2. Wu P, Wang Z, Zhu Q, Xie Z, Mei Y, Liang Y & Chen Z (2021) Stress preadaptation and overexpression of rpoS and hfq genes increase stress resistance of Pseudomonas fluorescens ATCC13525. Microbiological Research 250: 126804
    3.