Difference between revisions of "Part:BBa K2738006:Experience"
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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. | 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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<img scr="https://static.igem.org/mediawiki/2018/4/4d/T--Paris_Bettencourt--BB_006.png"> | <img scr="https://static.igem.org/mediawiki/2018/4/4d/T--Paris_Bettencourt--BB_006.png"> | ||
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3. Dynamic time-lapse imaging of bactericidal cell viability | 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. | 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="https://static.igem.org/mediawiki/2018/f/f6/T--Paris_Bettencourt--control_bsub.gif"> | + | Control: |
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| + | <img scr="https://static.igem.org/mediawiki/2018/f/f6/T--Paris_Bettencourt--control_bsub.gif"> | ||
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| − | Ovispirin+Ferritin: <img scr="https://static.igem.org/mediawiki/2018/4/44/T--Paris_Bettencourt--OV1_-_bsub.gif"> | + | Ovispirin+Ferritin: |
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| + | <img scr="https://static.igem.org/mediawiki/2018/4/44/T--Paris_Bettencourt--OV1_-_bsub.gif"> | ||
| + | </div> | ||
| + | 4. Biocompatibility towards mammalian cell membrane | ||
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<img scr="https://static.igem.org/mediawiki/2018/1/17/T--Paris_Bettencourt--liposome_c6.png"> | <img scr="https://static.igem.org/mediawiki/2018/1/17/T--Paris_Bettencourt--liposome_c6.png"> | ||
| + | </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. | ||
Latest revision as of 00:36, 18 October 2018
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Applications of BBa_K2738006
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="
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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="
">
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="
">
Ovispirin+Ferritin:
<img scr="
">
4. Biocompatibility towards mammalian cell membrane
<img scr="
">
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="">
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
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