Difference between revisions of "Team:Paris Bettencourt/BioBricks"

Line 96: Line 96:
 
<div class='textbody'>
 
<div class='textbody'>
 
<b>References:</b>
 
<b>References:</b>
−
<p>Ovispirin obtained from DRAMP database: DRAMP03826</br>
+
<p>1.Ovispirin obtained from DRAMP database: DRAMP03826</br>
−
<p>Ferritin obtained from RCSB. PDB number: 4XGS</br>
+
<p>2.Ferritin obtained from RCSB. PDB number: 4XGS</br>
−
<p>Star shaped antimicrobial peptides : 10.1038/NMICROBIOL.2016.162</p>   
+
<p>3.Star shaped antimicrobial peptides : 10.1038/NMICROBIOL.2016.162</p>   
 
</div>
 
</div>
 
</br>
 
</br>
 
</br>
 
</br>
 
</br>
 
</br>
 +
<div class='textbody h2'>
 +
<h2>Improved Part - Part: BBa_K2738104 (designed protein lyase cage-enterocin A)</h2>
 +
</div>
 +
<div class='textbody'>
 +
<p>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 protein-lyase-cage (self-assembling protein) to Enterocin A (antimicrobial peptide) via golden-gate assembly. The fusion protein expression is regulated by T7-promoter.</br>
 +
<p>The protein-lyase-cage is a multimeric, self-assembling protein formed by synthetically designed monomeric subunits. Enterocin A is an antimicrobial peptide, naturally produced by <i>Enterococcus faecium</i>.</p>
 +
</div>
 +
<div class='textbody h1'>
 +
<h1>Results</h1>
 +
</div>
 +
<div class='textbody h2'>
 +
<h2>Expression data</h2>
 +
</div>
 +
 +
<div class='textbody'>
 +
<p>The lyase-cage-enterocinA 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="text3 img"> 
 +
<img src="https://static.igem.org/mediawiki/2018/5/50/T--Paris_Bettencourt--BBa_2738104.png">
 +
</div>
 +
 +
<div class="textbody">
 +
<p>2. MIC and growth curve</br>
 +
<p>We determined the MIC of lyase-cage-enterocinA StarCore to inhibit the growth of <i>E. coli</i> and <i>B. subtilis</i> (figure A and B). We performed growth curve assay for <i>E. coli</i> and <i>B. subtilis</i> with and without antimicrobial peptides as shown in figure C and D.</br>
 +
<p>The results indicate that the lyase-cage-enterocinA StarCore has the MIC activity at 150 ug/ml for <i>B. subtilis</i> but not for <i>E. coli</i>. Moreover, at 7.3 uM it also affects the growth kinetics  <i>B. subtilis</i> to a significant extent indicating bactericidal activity. The antibacterial activity of lyase-cage-enterocinA StarCore is comparable to ovispirin at its MIC concentration (5 uM) alone as shown in the figure.</p>
 +
</div>
 +
 +
<div class="text3 img"> 
 +
<img src="https://static.igem.org/mediawiki/2018/a/a9/T--Paris_Bettencourt--BB_104.png">
 +
</div>
 +
 +
<div class='textbody'>
 +
<p>Biocompatibility towards mammalian cell membrane.</p>
 +
</div>
 +
<div class="text3 img"> 
 +
<img src="https://static.igem.org/mediawiki/2018/2/2d/T--Paris_Bettencourt--liposome_c104.png">
 +
</div>
 +
<div class="textbody span">
 +
<b>Figure 2.</b> Mammalian liposome leakage induced by lyase cage-Enterocin A StarCore (A) and the kinetics of liposome leakage (B). Ovispirin (5 uM) and lyase-ovispirin    (3.9 uM) are used at their MIC concentration obtained from previous results.
 +
</div>
 +
<div class="text3 img"> 
 +
<img src="https://static.igem.org/mediawiki/2018/2/2d/T--Paris_Bettencourt--liposome_c104.png">
 +
</div
 +
 +
<div class='textbody'>
 +
<p>Ovispirin alone lyses the liposome indicating damage to mammalian cell membrane. Lyase-cage-enterocin 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.</p>
 +
</div>
 +
<div class='textbody h2'>
 +
<h2>References</h2>
 +
</div>
 +
<div class='textbody'>
 +
<p>1.Protein lyase cage obtained from RCSB: 4QCC PDB</br> 
 +
<p>2.Enterocin reference from Bactibase: <a href="http://bactibase.hammamilab.org/BAC088>here</a></br>
 +
<p>3.Star shaped antimicrobial peptides : <a href="10.1038/NMICROBIOL.2016.162>here</a></br>
 +
<p>4.Liposome leakage assay: <a href="https://www.sciencedirect.com/science/article/pii/S007668799778025X">here</a></p>
 +
</div>
 +
 
 
   
 
   
 +
 +
  
  

Revision as of 01:49, 18 October 2018

BioBricks

New Part - Part: BBa_K2738006 (Ferritin-ovispirin)

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.

Figure 1: Ferritin-ovispirin-StarCore monomer and assembly obtained by homology modeling.

Results

1.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:

1.Ovispirin obtained from DRAMP database: DRAMP03826

2.Ferritin obtained from RCSB. PDB number: 4XGS

3.Star shaped antimicrobial peptides : 10.1038/NMICROBIOL.2016.162




Improved Part - Part: BBa_K2738104 (designed protein lyase cage-enterocin A)

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 protein-lyase-cage (self-assembling protein) to Enterocin A (antimicrobial peptide) via golden-gate assembly. The fusion protein expression is regulated by T7-promoter.

The protein-lyase-cage is a multimeric, self-assembling protein formed by synthetically designed monomeric subunits. Enterocin A is an antimicrobial peptide, naturally produced by Enterococcus faecium.

Results

Expression data

The lyase-cage-enterocinA fusion protein was produced via cell-free expression system. The expression was verified by SDS gel as shown in the figure below.

2. MIC and growth curve

We determined the MIC of lyase-cage-enterocinA 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 lyase-cage-enterocinA StarCore has the MIC activity at 150 ug/ml for B. subtilis but not for E. coli. Moreover, at 7.3 uM it also affects the growth kinetics B. subtilis to a significant extent indicating bactericidal activity. The antibacterial activity of lyase-cage-enterocinA StarCore is comparable to ovispirin at its MIC concentration (5 uM) alone as shown in the figure.

Biocompatibility towards mammalian cell membrane.

Figure 2. Mammalian liposome leakage induced by lyase cage-Enterocin A StarCore (A) and the kinetics of liposome leakage (B). Ovispirin (5 uM) and lyase-ovispirin (3.9 uM) are used at their MIC concentration obtained from previous results.

Ovispirin alone lyses the liposome indicating damage to mammalian cell membrane. Lyase-cage-enterocin 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

1.Protein lyase cage obtained from RCSB: 4QCC PDB

2.Enterocin reference from Bactibase: here

4.Liposome leakage assay: here

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
paris-bettencourt-2018@cri-paris.org