Difference between revisions of "Part:BBa K2043011"

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In the context of Paris Bettencourt 2016 iGEM project, Frank&Stain, we looked for fabric binding domains with phage display in order to optimise our enzymes on the topic of pigment degradation. More information can be consulted in the wiki, but we wanted to degrade wine stains, and we developed the FBD in order to direct the enzymatic activity to the fabric. <br><br>
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<p>
  
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We fused the GFP gene with the FBD2 (in the N-terminal of the GFP) in order to understand two things: (i)if the GFP fluorescence would be affect by the binding of the FBD and (ii)if the binding capacity would be affected. <br><br>
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This part corresponds to the GFP genes bound to the Fabric Binding Domain 2 (FBD2).
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In the context of Paris Bettencourt 2016 iGEM project, <a href="http://2016.igem.org/Team:Paris_Bettencourt">Frank&Stain</a>, we looked for novel fabric binding domains with phage display technique in order to optimise enzymes by fusing them with different FBD. <br>
 +
Results from our models suggest that the addition of an FBD with optimal affinity can concentrate an enzyme near the fabric surface and therefore increase stain-degrading enzymatic activity. Using the method of phage display, we isolated 40 short peptides with affinity for cotton, linen, wool, polyester or silk. Bioinformatics analysis identified sequence motifs and biophysical features associated with binding to each fabric, or non-specific binding to several fabrics. Several peptides were selected for further analysis by quantitative ELISA. The peptides discovered were used to fuse with GFP and stain-degrading enzymes to target wine-stained fabric samples. <br>
 +
The peptides are identified by in vitro phage-display screening of libraries containing typically 10<sup>9</sup> distinct, linear 7 amino-acid peptides against our target, in our case different fabrics - cotton, wool, silk, linen, polyester, and the library is enriched with the phages which binds to the fabric over several rounds of panning.<br><br>
  
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The results for FBD2-GFP are as follows:
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<b>Table 1</b> Characteristics of Fabric Binding Domain. The numbers next to each fabric means the number of times the sequence was repeated in each fabric<br>
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https://static.igem.org/mediawiki/parts/8/84/Paris_Bettencourt-biobricks_gfp2.png
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<table style="width:80%; border: 2px solid black;">
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<tr style="border: 2px solid black;">
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<th style="border: 2px solid black;"> Name </th>
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<th style="border: 2px solid black;"> Amino Acid Sequence </th>
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<th style="border: 2px solid black;"> Binding Affinity </th>
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<th style="border: 2px solid black;"> Charge </th>
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<th style="border: 2px solid black;"> Hydropathicity </th>
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</tr>
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<tr style="border: 2px solid black;">
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<td style="border: 2px solid black; text-align: center;"> FBD2 </th>
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<td style="border: 2px solid black; text-align: center;""> ADARYKS </th>
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<td style="border: 2px solid black; text-align: center;""> Nonspecific- Cotton: (5), Linen: (1), Polyester:(3) , Silk: (2), Wool: (9)</th>
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<td style="border: 2px solid black; text-align: center;""> +1 </th>
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<td style="border: 2px solid black; text-align: center;""> -1.486 </th>
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</tr>
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</table>
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<br>
 
<br>
 
<br>
  
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FBD-2 has a sequence of ADARYKS, is highly specific to vegetal-based fabrics cotton and linen in all tested conditions, with some specificity to wool, which is strongest in 5% BSA. This amino acid sequence has a positive charge, and a Hydropathicity (GRAVY) index of -1.486 (hydrophilic).
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<h2>Testing the part</h2>
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<br><br>
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These experiments were performed by obtaining cell extract of cells of a strain of <i>E. coli<(i> overexpressing FBD2-GFP, incubating the cell extract with the fabrics overnight, and then performing 2 washes  with the indicated solutions.
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ELISA for each FBD incubated with each fabric after two rounds of washing, normalized to the level after a single round. In figure 1 we see that FBD 2 binds only to Cotton and more strongly to linen.
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<br>
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<img src="https://static.igem.org/mediawiki/parts/a/a1/2016_paris_bettencourt_FBDs_fig_1.png" width=600><br>
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<img src="https://static.igem.org/mediawiki/parts/c/c2/2016_paris_bettencourt_FBDs_fig_2.png" width=600><br>
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<b>Figure 1</b> Fabric binding domain validation by ELISA. Control matrix repeating the procedure of panel A without phage.
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<br>
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<br>
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After doing this assay we fuse the FBD with GFP, which is actually the part we are submitting. Then we test the fluorescence of GFP to see if the fusion itself affects the activity or the expression.<br>
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<img src="https://static.igem.org/mediawiki/parts/5/5e/PRESENTATION.jpg" width=800><br>
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<b>Figure 2</b> Bar plot showing the fluorescence of cell extracts relative to the control (No GFP expressed). Here we compare the fluorescence of different cell extract expressing Fabric Binding Domains (FBDs) with affinity for different fabrics. The part <a href="https://parts.igem.org/Part:BBa_K1321357">BBa_K1321357</a> (GFP-CBDex) was used as positive control. The blue bar correspond with the negative control (no GFP in the cell extract) whereas the green ones with those whose fluorescence was higher than the control and in red those which presented no fluorescence, maybe because lacking activity or functionality.
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<br>
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<br>
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FBD2 didn’t pesent problems in activity as the figure 2 shows. The fluorescence of the cell extract was quiet strong even compared with the positive control CBD.
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<br>
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<br>
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Once we observed that this fusion protein worked properly, we assayed the strength of the binding in different fabrics by adding cell extract in a 96 wells plate which contains the pieces of fabric inside. The cell extract was washed with different solutions and the final fluorescence was measured after two washes.
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<br>
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<img src="https://static.igem.org/mediawiki/parts/1/1d/FBD2.jpg" width=600><br>
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<b>Figure 3</b>Cell extract from E. coli expressing this part was incubated overnight in a 96 well plate with fabric placed at the bottom of the wells. Water, PBS, 5% BSA, 70% Ethanol and Catechol 30mM. The data displayed corresponds to the values after the final wash, normalised using the values from the first wash. The intensity of the colour corresponds to the GFP signal measured at that point. Excitation 475nm and emission 515nm.<br>
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<br>
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According with the figure above, the results coincide with the ELISA analysis, since it shows certain specifity for cotton and linen. Both cellulosic fabrics.<br><br><br>
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<h2>References</h2>
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General In Vitro Method to Analyze the Interactions of Synthetic Polymers with Human Antibody Repertoires,Anandakumar Soshee, Stefan Zürcher, Nicholas D. Spencer, Avraham Halperin, and Clément Nizak,Biomacromolecules 2014 15 (1), 113-121, DOI: 10.1021/bm401360y
 
<br><br>
 
<br><br>
 +
Hierarchy and extremes in selections from pools of randomized proteins,PNAS 2016 113 (13) 3482-3487; published ahead of print March 11, 2016,doi:10.1073/pnas.1517813113
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<br><br>
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Identification of Soft Matter Binding Peptide Ligands Using Phage Display Kemal Arda Gü nay and Harm-Anton Klok, Bioconjugate Chem. 2015, 26, 2002−2015, DOI: 10.1021/acs.bioconjchem.5b00377
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<br><br>
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Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis, Binétruy-Tournaire R1, Demangel C, Malavaud B, Vassy R, Rouyre S, Kraemer M, Plouët J, Derbin C,Perret G, Mazié JC, EMBO J. 2000 Apr 3;19(7):1525-33
 +
<br><br>
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Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains Hennie R.Hoogenboom1 , Andrew D.Griffiths1 , Kevin S.Johnson2 , David J.Chiswell2 , Peter Hudson4 and Greg Winter, 1991 Nucleic Acids Research, Vol. 19, No. 15 4133-4137.
 +
<br><br>
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Random Peptide Libraries: A Source of Specific Protein Binding Molecules , SCIENCE, VOL. 249, JAMES J. DEVLIN,* Lucy C. PANGANIBAN, PATRICIA E. DEVLIN, 1999
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<br><br>
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Selection of Arginine-Rich Anti-Gold Antibodies Engineered for Plasmonic Colloid Self-Assembly,Purvi Jain, Anandakumar Soshee, S. Shankara Narayanan, Jadab Sharma, Christian Girard, Erik Dujardin, and Clément Nizak, The Journal of Physical Chemistry C 2014 118 (26), 14502-14510, DOI: 10.1021/jp502118n
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<br><br>
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Selection of phage display combinatorial library peptides with affinity for a yohimbine imprinted methacrylate polymer, Johanna Berglunda, Christer Lindbladha, Ian A. Nichollsb and Klaus Mosbach, Analytical Communications, January 1998, Vol 35 (3–7)
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<br><br>
 +
Self-assembled artificial viral capsid decorated with gold nanoparticles, Kazunori Matsuura, Genki Ueno and Seiya Fujita, Polymer Journal 47, 146-151 (February 2015) | doi:10.1038/pj.2014.99
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</p>
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</html>
  
 
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<partinfo>BBa_K2043011 parameters</partinfo>
 
<partinfo>BBa_K2043011 parameters</partinfo>
 
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<h1>GFP-FBD2</h1>
 
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Jain, P., Soshee, A., Narayanan, S. S., Sharma, J., Girard, C., Dujardin, E., & Nizak, C. (2014). Selection of arginine-rich anti-gold antibodies engineered for plasmonic colloid self-assembly. The Journal of Physical Chemistry C, 118(26), 14502-14510.<br><br>
 
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Soshee, A., Zürcher, S., Spencer, N. D., Halperin, A., & Nizak, C. (2013). General in vitro method to analyze the interactions of synthetic polymers with human antibody repertoires. Biomacromolecules, 15(1), 113-121.<br><br>
 
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Boyer, S., Biswas, D., Soshee, A. K., Scaramozzino, N., Nizak, C., & Rivoire, O. (2016). Hierarchy and extremes in selections from pools of randomized proteins. Proceedings of the National Academy of Sciences, 201517813.<br><br>
 

Revision as of 02:44, 28 October 2016


GFP-FBD2


This part corresponds to the GFP genes bound to the Fabric Binding Domain 2 (FBD2). In the context of Paris Bettencourt 2016 iGEM project, Frank&Stain, we looked for novel fabric binding domains with phage display technique in order to optimise enzymes by fusing them with different FBD.
Results from our models suggest that the addition of an FBD with optimal affinity can concentrate an enzyme near the fabric surface and therefore increase stain-degrading enzymatic activity. Using the method of phage display, we isolated 40 short peptides with affinity for cotton, linen, wool, polyester or silk. Bioinformatics analysis identified sequence motifs and biophysical features associated with binding to each fabric, or non-specific binding to several fabrics. Several peptides were selected for further analysis by quantitative ELISA. The peptides discovered were used to fuse with GFP and stain-degrading enzymes to target wine-stained fabric samples.
The peptides are identified by in vitro phage-display screening of libraries containing typically 109 distinct, linear 7 amino-acid peptides against our target, in our case different fabrics - cotton, wool, silk, linen, polyester, and the library is enriched with the phages which binds to the fabric over several rounds of panning.

Table 1 Characteristics of Fabric Binding Domain. The numbers next to each fabric means the number of times the sequence was repeated in each fabric

Name Amino Acid Sequence Binding Affinity Charge Hydropathicity
FBD2 ADARYKS Nonspecific- Cotton: (5), Linen: (1), Polyester:(3) , Silk: (2), Wool: (9) +1 -1.486


Testing the part

ELISA for each FBD incubated with each fabric after two rounds of washing, normalized to the level after a single round. In figure 1 we see that FBD 2 binds only to Cotton and more strongly to linen.


Figure 1 Fabric binding domain validation by ELISA. Control matrix repeating the procedure of panel A without phage.

After doing this assay we fuse the FBD with GFP, which is actually the part we are submitting. Then we test the fluorescence of GFP to see if the fusion itself affects the activity or the expression.

Figure 2 Bar plot showing the fluorescence of cell extracts relative to the control (No GFP expressed). Here we compare the fluorescence of different cell extract expressing Fabric Binding Domains (FBDs) with affinity for different fabrics. The part BBa_K1321357 (GFP-CBDex) was used as positive control. The blue bar correspond with the negative control (no GFP in the cell extract) whereas the green ones with those whose fluorescence was higher than the control and in red those which presented no fluorescence, maybe because lacking activity or functionality.

FBD2 didn’t pesent problems in activity as the figure 2 shows. The fluorescence of the cell extract was quiet strong even compared with the positive control CBD.

Once we observed that this fusion protein worked properly, we assayed the strength of the binding in different fabrics by adding cell extract in a 96 wells plate which contains the pieces of fabric inside. The cell extract was washed with different solutions and the final fluorescence was measured after two washes.

Figure 3Cell extract from E. coli expressing this part was incubated overnight in a 96 well plate with fabric placed at the bottom of the wells. Water, PBS, 5% BSA, 70% Ethanol and Catechol 30mM. The data displayed corresponds to the values after the final wash, normalised using the values from the first wash. The intensity of the colour corresponds to the GFP signal measured at that point. Excitation 475nm and emission 515nm.

According with the figure above, the results coincide with the ELISA analysis, since it shows certain specifity for cotton and linen. Both cellulosic fabrics.


References

General In Vitro Method to Analyze the Interactions of Synthetic Polymers with Human Antibody Repertoires,Anandakumar Soshee, Stefan Zürcher, Nicholas D. Spencer, Avraham Halperin, and Clément Nizak,Biomacromolecules 2014 15 (1), 113-121, DOI: 10.1021/bm401360y

Hierarchy and extremes in selections from pools of randomized proteins,PNAS 2016 113 (13) 3482-3487; published ahead of print March 11, 2016,doi:10.1073/pnas.1517813113

Identification of Soft Matter Binding Peptide Ligands Using Phage Display Kemal Arda Gü nay and Harm-Anton Klok, Bioconjugate Chem. 2015, 26, 2002−2015, DOI: 10.1021/acs.bioconjchem.5b00377

Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis, Binétruy-Tournaire R1, Demangel C, Malavaud B, Vassy R, Rouyre S, Kraemer M, Plouët J, Derbin C,Perret G, Mazié JC, EMBO J. 2000 Apr 3;19(7):1525-33

Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains Hennie R.Hoogenboom1 , Andrew D.Griffiths1 , Kevin S.Johnson2 , David J.Chiswell2 , Peter Hudson4 and Greg Winter, 1991 Nucleic Acids Research, Vol. 19, No. 15 4133-4137.

Random Peptide Libraries: A Source of Specific Protein Binding Molecules , SCIENCE, VOL. 249, JAMES J. DEVLIN,* Lucy C. PANGANIBAN, PATRICIA E. DEVLIN, 1999

Selection of Arginine-Rich Anti-Gold Antibodies Engineered for Plasmonic Colloid Self-Assembly,Purvi Jain, Anandakumar Soshee, S. Shankara Narayanan, Jadab Sharma, Christian Girard, Erik Dujardin, and Clément Nizak, The Journal of Physical Chemistry C 2014 118 (26), 14502-14510, DOI: 10.1021/jp502118n

Selection of phage display combinatorial library peptides with affinity for a yohimbine imprinted methacrylate polymer, Johanna Berglunda, Christer Lindbladha, Ian A. Nichollsb and Klaus Mosbach, Analytical Communications, January 1998, Vol 35 (3–7)

Self-assembled artificial viral capsid decorated with gold nanoparticles, Kazunori Matsuura, Genki Ueno and Seiya Fujita, Polymer Journal 47, 146-151 (February 2015) | doi:10.1038/pj.2014.99

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]