Part:BBa_K2273040

Part Information
RFC standard	RFC 25
Fused Tag	BBa_K2273017: mini. SpyCatcher His-tagged
Fluorescent Protein	BBa_K2273033: sfGFP
Submitted by	[http://2017.igem.org/Team:TU_Dresden TU Dresden]

sfGFP with C-Terminal SpyCatcher and His Tag

This composite part was used for evalutaion in the [http://2017.igem.org/Team:TU_Dresden/Project/Secretion secretion project] of 2017 TU_Dresden iGEM [http://2017.igem.org/Team:TU_Dresden team]. It codes for a fluorescent reporter protein (sfGFP) and a functional tag (mini. SpyCatcher), mediating covalent bonding with it tag partner (SpyTag). It is optimized for usage in Bacillus subtilis.

Design

A signal peptide (AmyE SP) was fused n-terminally to this construct to induce secretion.

Figure 1“: Genetic constructs with sfGFP. Depicted are translational fusion constructs downstream of the PxylA promotor, that were cloned in the multiple cloning site of the pBS2EPxylA vector. The constructs contain a signal peptide sequence, the gene coding for mCherry, either c- or n-terminally fused SpyTag or mini. SpyCatcher and a his-tag.

Application

The successful secretion could be proven with a fluorescence assay using the supernatants of B. subtilis (Figure 2 and Figure 3)

Figure 2“: Endpoint measurement of the fluorescence from supernatants carrying our constructs and the wild type. Expression of the single copy mCherry or sfGFP fusion SpyTag/SpyCather 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 for sfGFP was set to 480 nm and emission was recorded at 510 nm and for mCherry excitation wavelength was set to 585 nm and emission was recorded at 615 nm . The fluorescence was normalized by the optical density (OD600). Graph shows mean values and standard deviations of at least two biological and three technical replicates.

Figure 3“ Supernatants of B. subtilis cultures excited with blue light. Wild-type supernatant (left) and a SpyTag-sfGFP secreting strain (middle and right). The expression of the multi-copy sfGFP was induced with 1% Xylose and the supernatant was harvested after 16 h of incubation. The fluorescence was induced with a “Dark Reader Transilluminator”.

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]