Part:BBa_K1989004

Triplespytag with mRFP and His-tag

Usage and Biology

In the last few years, hydrogens made from natural or synthetic polymers have been investigated due to their extensive application in clinical medicine and synthetic biology. Compared to traditional biological material, protein-based multifunctional biological material is low-cost, facile and eco-friendly. However, strategies for assembling 3D molecular networks synthesized only by protein molecular remain underdeveloped. The reason why investigating this technology is still tough is lack of protein-based cross linking agents. Inspired by the self-catalysis of isopeptide bond between Lys and Asp in Streptococcus pyogenes fibronectin-binding protein FbaB, researchers split the catalytic domain and obtained two peptide called SpyTag(the short one) and SpyCatcher(the long one) which are able to form isopeptide bond with the other without any assistant. By fusing SpyTag and SpyCatcher with functional domains respectively, researchers solved the problem tactfully. In order to using SpyTag and SpyCatcher system as scaffold, we fused three SpyTag spaced by (VPGVG)4 with 6xHistag in N-terminal and another functional protein called mRFP in C-terminal. This functional protein is obtained from BBa_E1010.

Based on our results, the fused protein His-triple SpyTag-mRFP (His-3A-Red) possess both isopeptide bond forming function and coloration ability. Thus, using 3A-Red as a part of hydrogel formation, we could obtain visible hydrogel.

You can find triplespycatcher here https://parts.igem.org/Part:BBa_K1989001".

Cultivation, Purification and SDS-PAGE

Cultivation

The part was assembled with T7 promoter and RBS in pET28a plasmid vector. E. coli strain BL21(DE3) harboring the appropriate plasmid was grown at 37 °C in 2xYT medium overnight with suitable concentration of antibiotic. The culture was diluted 100 fold into fresh medium with antibiotic and grown at 37°C to an optical density of 0.6~0.8 at 600 nm, the protein expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and cells were grown overnight at 25°C.

Purification

Cells were centrifuged at 8000rpm for 15min at 4°C. Resuspend the cell paste expressing recombinant protein in binding buffer (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1mM β-mercaptoethanol, pH7.4), containing SIGMAFAST™ Protease Inhibitor Cocktail Tablets (SIGMA-ALORICH). Disrupt the cells with sonication for 20 min with suitable power on ice and centrifuge at 18000 rpm for 40 min at 4°C. Remove remaining particles by passing the supernatant through a 0.22 μm filter. The HisTrap™ column (GE Healthcare, Inc.) was equilibrated with binding buffer. Load the sample and wash the column with binding buffer.

Elute the target protein with a linear gradient starting with binding buffer and ending with the same buffer including 500mM imidazole. The eluted fraction containing the target protein were concentrated by Amicon® Ultra Centrifugal Filters (Merck) with a 10 kDa cutoff, then frozen by liquid nitrogen and stored at -80°C.

SDS-PAGE

Protein purification was checked by SDS-PAGE and the resulting protein is quantified by Braford analysis.

References

1. Rodolphe Barrangou, Christophe Fremaux, Hélène Deveau, et al. CRISPR provides acquired resistance against viruses. Science, 2007, 315: 1709-1712.

2. Deltcheva E, Chylinski K, Sharma CM, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 2011;471:602–7.

3. Martin Jinek, Krzysztof Chylinski, Ines Fonfara, et al. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 2012, 337: 816-821.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 959
  Illegal AgeI site found at 1071
  
• 1000
  COMPATIBLE WITH RFC[1000]