Difference between revisions of "Part:BBa K2738006"

 
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2738006 short</partinfo>
 
<partinfo>BBa_K2738006 short</partinfo>
  
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Ovispirin is an AMP
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<p>
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Ferritin is iron chelating multimeric complex protein
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Part: BBa_K2738006 (Ferritin-ovispirin)
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Description
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<li> We aimed to fuse antimicrobial peptides to self-assembling multimeric proteins giving us star-shape antimicrobial peptides called StarCores. The StarCore described here is a result of the fusion of Ferritin (self-assembling protein) to ovispirin (antimicrobial peptide) via golden-gate assembly. The fusion protein expression is regulated by T7-promoter. </li>
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<li> Ferritin is a universal intracellular protein produced by almost all living organisms, including algae, bacteria, higher plants, and animals. Ferritin is a globular protein complex consisting of 24 protein subunits forming a nanocage. Ovispirin is an alpha-helical antimicrobial peptide derived from derived SMAP29 peptide.It was found to inhibit several antibiotic-resistant bacterial strains. </li>
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<li> Using homology modelling, we obtained the structure of ferritin-ovispirin fusion monomer protein. Using homology alignment, we were able to reconstruct the assembled ferritin-ovispirin monomer into the multimeric star-shaped construct. </li>
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<p> 
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https://static.igem.org/mediawiki/2018/f/f8/T--Paris_Bettencourt--c6_monomer.gif
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Monomer
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</p>
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<p>
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https://static.igem.org/mediawiki/2018/f/fc/T--Paris_Bettencourt--c6_assemble.gif
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Assembly
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</p>
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<p>
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Results:
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Expression verification
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The ferritin-ovispirin fusion protein was produced via cell-free expression system. The expression was verified by SDS gel as shown in the figure below. </p>
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<div class="text1 img">
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<img src="https://static.igem.org/mediawiki/2018/f/fc/T--Paris_Bettencourt--BBa_K2738006.png ">
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Figure 2. Cell free expression and purification of Ferritin-ovispirin-StarCore
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</div>
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<p>
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2. MIC and growth curve
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We determined the MIC of ferritin-ovispirin StarCore to inhibit the growth of E coli and B subtilis (figure A and B). We performed growth curve assay for E coli and B subtilis with and without antimicrobial peptides as shown in figure C and D. 
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</p>
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<p>
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The results indicate that the ferritin-ovispirin StarCore has the MIC activity at 3 ug/ml for both E coli and B subtilis. Moreover, at 7.3 uM it also affects their growth kinetics to a significant extent indicating bactericidal activity. The antibacterial activity of ferritin-ovispirin StarCore is comparable to ovispirin at its MIC concentration (5 uM) alone as shown in the figure.
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</p>
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<div class="text1 img">
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<img src="https://static.igem.org/mediawiki/2018/4/4d/T--Paris_Bettencourt--BB_006.png">
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</div>
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<p>
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3. Dynamic time-lapse imaging of bactericidal cell viability
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The following results were obtained by time-lapse microscopy imaging, to study the bactericidal effect of antimicrobial peptides on B subtilis viability. Here we can observe that the presence of antimicrobial peptide proves to be toxic for the growing B subtilis which supports our growth curve data and MIC data. 
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</p>
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Control:
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<div class="text1 img">
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<img src="https://static.igem.org/mediawiki/2018/f/f6/T--Paris_Bettencourt--control_bsub.gif">
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</div>
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Ovispirin+Ferritin:
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<div class="text1 img">
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<img src="https://static.igem.org/mediawiki/2018/4/44/T--Paris_Bettencourt--OV1_-_bsub.gif">
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</div>
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4. Biocompatibility towards mammalian cell membrane
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<div class="text1 img">
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<img src="https://static.igem.org/mediawiki/2018/1/17/T--Paris_Bettencourt--liposome_c6.png">
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</div>
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Figure 1. Mammalian liposome leakage induced by ferritin-ovispirin StarCore (A) and the kinetics of liposome leakage (B). Ovispirin (5 uM) and ferritin-ovispirin (3 uM) are used at their MIC concentration obtained from previous results.
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<img src="https://static.igem.org/mediawiki/2018/1/17/T--Paris_Bettencourt--liposome_c6.png">
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To test the biocompatibility of the StarCores with mammalian membrane, we performed liposome leakage assay. Ovispirin alone lyses the liposome indicating damage to mammalian cell membrane. Ferritin-ovispirin has a reduced effect on the liposome leakage indicating an improvement in biocompatibility towards mammalian cell membrane. The Starcore ferritin-ovispirin has a safer profile in terms of membrane activity as compared to ovispirin alone at their respective MIC concentration.
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References:
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Ovispirin obtained from DRAMP database: DRAMP03826
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Ferritin obtained from RCSB. PDB number: 4XGS
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Star shaped antimicrobial peptides : 10.1038/NMICROBIOL.2016.162 
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 13:19, 19 November 2018

__NOTOC__ BBa_K2738006 short

Part: BBa_K2738006 (Ferritin-ovispirin) Description

  • We aimed to fuse antimicrobial peptides to self-assembling multimeric proteins giving us star-shape antimicrobial peptides called StarCores. The StarCore described here is a result of the fusion of Ferritin (self-assembling protein) to ovispirin (antimicrobial peptide) via golden-gate assembly. The fusion protein expression is regulated by T7-promoter.
  • Ferritin is a universal intracellular protein produced by almost all living organisms, including algae, bacteria, higher plants, and animals. Ferritin is a globular protein complex consisting of 24 protein subunits forming a nanocage. Ovispirin is an alpha-helical antimicrobial peptide derived from derived SMAP29 peptide.It was found to inhibit several antibiotic-resistant bacterial strains.
  • Using homology modelling, we obtained the structure of ferritin-ovispirin fusion monomer protein. Using homology alignment, we were able to reconstruct the assembled ferritin-ovispirin monomer into the multimeric star-shaped construct.

    • https://static.igem.org/mediawiki/2018/f/f8/T--Paris_Bettencourt--c6_monomer.gif Monomer

    https://static.igem.org/mediawiki/2018/f/fc/T--Paris_Bettencourt--c6_assemble.gif Assembly

    Results: Expression verification The ferritin-ovispirin fusion protein was produced via cell-free expression system. The expression was verified by SDS gel as shown in the figure below.

    Figure 2. Cell free expression and purification of Ferritin-ovispirin-StarCore

    2. MIC and growth curve We determined the MIC of ferritin-ovispirin StarCore to inhibit the growth of E coli and B subtilis (figure A and B). We performed growth curve assay for E coli and B subtilis with and without antimicrobial peptides as shown in figure C and D.

    The results indicate that the ferritin-ovispirin StarCore has the MIC activity at 3 ug/ml for both E coli and B subtilis. Moreover, at 7.3 uM it also affects their growth kinetics to a significant extent indicating bactericidal activity. The antibacterial activity of ferritin-ovispirin StarCore is comparable to ovispirin at its MIC concentration (5 uM) alone as shown in the figure.

    3. Dynamic time-lapse imaging of bactericidal cell viability The following results were obtained by time-lapse microscopy imaging, to study the bactericidal effect of antimicrobial peptides on B subtilis viability. Here we can observe that the presence of antimicrobial peptide proves to be toxic for the growing B subtilis which supports our growth curve data and MIC data.

    Control:
    Ovispirin+Ferritin:
    4. Biocompatibility towards mammalian cell membrane
    Figure 1. Mammalian liposome leakage induced by ferritin-ovispirin StarCore (A) and the kinetics of liposome leakage (B). Ovispirin (5 uM) and ferritin-ovispirin (3 uM) are used at their MIC concentration obtained from previous results. To test the biocompatibility of the StarCores with mammalian membrane, we performed liposome leakage assay. Ovispirin alone lyses the liposome indicating damage to mammalian cell membrane. Ferritin-ovispirin has a reduced effect on the liposome leakage indicating an improvement in biocompatibility towards mammalian cell membrane. The Starcore ferritin-ovispirin has a safer profile in terms of membrane activity as compared to ovispirin alone at their respective MIC concentration. References: Ovispirin obtained from DRAMP database: DRAMP03826 Ferritin obtained from RCSB. PDB number: 4XGS Star shaped antimicrobial peptides : 10.1038/NMICROBIOL.2016.162 Sequence and Features BBa_K2738006 SequenceAndFeatures