Difference between revisions of "Part:BBa K2738006"
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Assembly
 
Assembly
  
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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. 
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<img scr="https://static.igem.org/mediawiki/2018/6/6d/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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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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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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<div class="hub_icon">
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<img scr="https://static.igem.org/mediawiki/2018/4/4d/T--Paris_Bettencourt--BB_006.png">
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</div>
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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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Control:
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<div class="textbody img">
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<img scr="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="textbody img">
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<img scr="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="textbody img">
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<img scr="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 scr="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 
  
  

Revision as of 03:37, 18 October 2018


Ovispirin fused to N-ter of Ferritin protein 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. <img scr="T--Paris_Bettencourt--c6_monomer.gif"> Monomer

<img scr="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. <img scr="T--Paris_Bettencourt--BBa_K2738006.png">

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.

<img scr="T--Paris_Bettencourt--BB_006.png">

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:

<img scr="T--Paris_Bettencourt--control_bsub.gif">

Ovispirin+Ferritin:

<img scr="T--Paris_Bettencourt--OV1_-_bsub.gif">

4. Biocompatibility towards mammalian cell membrane

<img scr="T--Paris_Bettencourt--liposome_c6.png">

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. <img scr="T--Paris_Bettencourt--liposome_c6.png">

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


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]