Part:BBa_K2273015

Part Information
RFC standard	RFC 25
Partner Tag	BBa_K2273014: SpyTag
Original Biobrick Part	BBa_K1159200: SpyCatcher
His-tagged mini.SpyCatcher	BBa_K2273017: mini.SpyCatcher-His
Submitted by	[http://2017.igem.org/Team:TU_Dresden TU Dresden]

Minimal SpyCatcher codon adapted for Bacillus subtilis

This is a shortened and codon adapted version of the SpyCatcher improved for secretion in B. subtilis. It codes for a part of the fibronectin-binding protein FbaB from Streptococcus pyogenes that forms a covalent isopeptide bond with a short oligopeptide called SpyTag. There is the original (BBa_K1159201) and the codon adapted version (BBa_K2273014) of this oligopeptide in the registry. The SpyTag and the Minimal SpyCatcher can be fused as a Tag to proteins of interest and will then mediate covalent bonding of these proteins.

Improvement

Minimization:

The SpyCatcher is a highly functional Tag, that can interact with its protein partner: the SpyTag. Upon interaction, additional C- or N-terminally translational fused proteins are brought in close proximity and can be linked together via this Tag system. Unfortunately the size of the SypCather (116 amino acid) is quite large and can therefore hinder the native folding of a linked the protein of interest.The 2017 team of [http://2017.igem.org/Team:TU_Dresden TU_Dresden] submitted a minimized version of the SpyCatcher to the registry to ensure the effective [http://2017.igem.org/Team:TU_Dresden/Project/Secretion secretion] necessary for their project. The mini. SpyCatcher is 32 amino acids shorter but still fully functional, which was proven by TU_Dresden team (see Application) as well as by the researchersthat proposed the minimization.

Codon adaption:

The original part was used in E. coli but the mini. SpyCatcher was thought to be implemented throughout TU_Dresdens project in B. subtilis. Therefore the sequence was codon adapted for this purposes. The sequence was proposed by researchersthat investigated the secretion of SpyTag/SpyCatcher system in B. subtilis.

His-Tag:

The mini. SpyCatcher was also submitted to the registry with an added C-terminal His-Tag to purify the protein constructs when SpyTag/SpyCatcher link together.

Application

The 2017 team of [http://2017.igem.org/Team:TU_Dresden/Project/Secretion TU_Dresden] used mini. SpyCatcher together with its partner SpyTag to investigate the potential of using B. subtilis secretion system for the production of self conjugation multi protein complexes. The his-tagged mini. SpyCatcher was C-terminally fused to mCherry which functions as a reporter protein. The secretion was mediated through the N-terminal signal peptide of AmyE. The secretion of the functional mCherry fusion protein could be proven (Figure 1 and Figure 2). The functionality of the mini. SpyCatcher was proven through SDS-Page (Figure 3). After incubating supernatants containing mCherry-mini. SpyCatcher and mCherry-SpyTag the fusion product was detected, proving the covalent bonding of the Tag partners.

Figure 1: Endpoint measurement of the fluorescence from supernatants. Expression of the multi copy mCherry constructs (purple) was induced with 1% Xylose and the supernatants were harvested after 16 h of incubation. Wild type supernatant is shown as a control (pink). Excitation wavelength was set to 585 nm and emission was recorded at 615 nm. The fluorecense was normalized over the optical density of the cell culture at 600 nm (OD600). Graph shows mean values and standard deviations of at least two biological and three technical replicates.

Figure 2:Supernatants of B. subtilis cultures. Wild-type supernatant (left) and a mCherry-mini. SpyCatcher secreting strain (right). The expression of the multi-copy mCherry was induced with 1% Xylose and the supernatant was harvested after 16 h of incubation.

Figure 3: SDS gel with crude and purified supernatants. Expression of the multi copy mCherry constructs was induced with 1% Xylose and the supernatants were harvested after 16 h of incubation. The his-tagged proteins were purified with Ni-NTA agarose beads. Lane 1 was loaded with 3 µl of NEB´s “Color Prestained Protein Standard Broad Range” ladder. Crude (c) and purified (p) supernatant of wild-type (WT) are shown as a control in lane 2 and 3. Lane 4 and 5 contain the supernatant of B. subtilis producing mCherry-mini. SpyCatcher fusion protein (36,6 kDa). Lane 4 and 5 contain the supernatant of B. subtilis producing mCherry-SpyTag fusion protein (31,9 kDa). The crude supernatants of the two mCherry producing strains were combined, incubated for 4 h, purified and loaded onto lane 8 and 9. The fusion product of the mCherry constructs is visable in the crude and purified supernatant.

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]