Part:BBa_K2043016

GFP-FBD9

Introduction

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 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. Bioinformatic analysis identified sequence motifs and biophysical features associated with binding to each fabric, or nonspecific 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
FBD9	FRKKRKS	Specific to linen: Cotton(0), Linen(1), Polyester(0), Silk(0), Wool(0)	+5	-2.671

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 9 is seen to be able to bind with range of affinities for different fabrics, so it was chosen to perform further experiments to find the dissociation constant (Kd).


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


Figure 2 ELISA signal determined afer multple rounds of washing. Points represent individual measurements from three replicates. Lines represent best-fit to an exponential decay model.

Using mass action reaction, the Kd for FBD9 binding on cotton was found to be 2x10^-5M +/- 1x10^^-5M which matches with our results from modelling.
After doing this assay we fuse the FBD9 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 3 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.

FBD9 didn’t pesent problems in activity as the figure 2 shows. The fluorescence of the cell extract was quiet strong even compared with the control.
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 4 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.

According with the figure above, FBD9 presents spicificity by silk and linen when it is washed with ethanol. Whereas in the ELISA analysis it only shows affinity by wool.

References

Berglund, J., Lindbladh, C., Mosbach, K., & Nicholls, I. A. (1998). Selection of phage display combinatorial library peptides with affinity for a yohimbine imprinted methacrylate polymer. Analytical Communications, 35(1), 3-7.

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.

Devlin, J. J., Panganiban, L. C., & Devlin, P. E. (1990). Random peptide libraries: a source of specific protein binding molecules. Science, 249(4967), 404-406.

Goncalves, V., Gautier, B., Garbay, C., Vidal, M., & Inguimbert, N. (2007). Development of a chemiluminescent screening assay for detection of vascular endothelial growth factor receptor 1 ligands. Analytical biochemistry, 366(1), 108-110.

Günay, K. A., & Klok, H. A. (2015). Identification of Soft Matter Binding Peptide Ligands Using Phage Display. Bioconjugate chemistry, 26(10), 2002-2015.

Hoogenboom, H. R., Griffiths, A. D., Johnson, K. S., Chiswell, D. J., Hudson, P., & Winter, G. (1991). Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic acids research, 19(15), 4133-4137.

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.

Matsuura, K., Ueno, G., & Fujita, S. (2015). Self-assembled artificial viral capsid decorated with gold nanoparticles. Polymer Journal, 47(2), 146-151.

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.

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]