Difference between revisions of "Part:BBa K2738006"
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Part: BBa_K2738006 (Ferritin-ovispirin) | Part: BBa_K2738006 (Ferritin-ovispirin) | ||
Description | 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. | + | <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> |
| − | 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> 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> |
| − | 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> 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> |
| − | < | + | |
| + | <p> | ||
| + | https://static.igem.org/mediawiki/2018/f/f8/T--Paris_Bettencourt--c6_monomer.gif | ||
Monomer | Monomer | ||
| + | </p> | ||
| − | < | + | <p> |
| + | https://static.igem.org/mediawiki/2018/f/fc/T--Paris_Bettencourt--c6_assemble.gif | ||
Assembly | Assembly | ||
| + | </p> | ||
| + | <p> | ||
Results: | Results: | ||
Expression verification | 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. | + | 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> |
| − | <div class=" | + | |
| − | <img | + | <div class="text1 img"> |
| + | <img src="https://static.igem.org/mediawiki/2018/f/fc/T--Paris_Bettencourt--BBa_K2738006.png "> | ||
Figure 2. Cell free expression and purification of Ferritin-ovispirin-StarCore | Figure 2. Cell free expression and purification of Ferritin-ovispirin-StarCore | ||
</div> | </div> | ||
| − | + | <p> | |
2. MIC and growth curve | 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. | 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. | ||
| + | </p> | ||
| − | 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. | + | <p> |
| − | <div class=" | + | 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 | + | </p> |
| + | |||
| + | <div class="text1 img"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/4/4d/T--Paris_Bettencourt--BB_006.png"> | ||
</div> | </div> | ||
| + | <p> | ||
3. Dynamic time-lapse imaging of bactericidal cell viability | 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. | 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. | ||
| + | </p> | ||
Control: | Control: | ||
| − | <div class=" | + | <div class="text1 img"> |
| − | <img | + | <img src="https://static.igem.org/mediawiki/2018/f/f6/T--Paris_Bettencourt--control_bsub.gif"> |
</div> | </div> | ||
Ovispirin+Ferritin: | Ovispirin+Ferritin: | ||
| − | <div class=" | + | <div class="text1 img"> |
| − | <img | + | <img src="https://static.igem.org/mediawiki/2018/4/44/T--Paris_Bettencourt--OV1_-_bsub.gif"> |
</div> | </div> | ||
4. Biocompatibility towards mammalian cell membrane | 4. Biocompatibility towards mammalian cell membrane | ||
| − | <div class=" | + | <div class="text1 img"> |
| − | <img | + | <img src="https://static.igem.org/mediawiki/2018/1/17/T--Paris_Bettencourt--liposome_c6.png"> |
</div> | </div> | ||
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. | 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 | + | <img src="https://static.igem.org/mediawiki/2018/1/17/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. | 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. | ||
Latest revision as of 13:19, 19 November 2018
__NOTOC__
Part: BBa_K2738006 (Ferritin-ovispirin) Description
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:
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