Part:BBa_K525406

Fusion Protein of S-Layer SbpA and mCerulean

Fusion protein of S-layer SgsE and mCerulean.

S-layers (crystalline bacterial surface layer) are crystal-like layers consisting of multiple protein monomers and can be found in various (archae-)bacteria. They constitute the outermost part of the cell wall. Especially their ability for self-assembly into distinct geometries is of scientific interest. At phase boundaries, in solutions and on a variety of surfaces they form different lattice structures. The geometry and arrangement is determined by the C-terminal self assembly-domain, which is specific for each S-layer protein. The most common lattice geometries are oblique, square and hexagonal. By modifying the characteristics of the S-layer through combination with functional groups and protein domains as well as their defined position and orientation to eachother (determined by the S-layer geometry) it is possible to realize various practical applications ([http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2006.00573.x/full Sleytr et al., 2007]).

Usage and Biology

S-layer proteins can be used as scaffold for nanobiotechnological applications and devices by e.g. fusing the S-layer's self-assembly domain to other functional protein domains. It is possible to coat surfaces and liposomes with S-layers. A big advantage of S-layers: after expressing in E. coli and purification, the nanobiotechnological system is cell-free. This enhances the biological security of a device.

This fluorescent S-layer fusion protein is used to characterize purification methods and the S-layer's ability to self-assemble on surfaces. It is also possible to use the characteristic of mCerulean as a pH indicator or as FRET donor ([http://pubs.acs.org/doi/abs/10.1021/bm901071b Kainz et al., 2010]).

Important parameters

Experiment	Characteristic	Result
Expression (E. coli)	Localisation	Inclusion body
	Compatibility	E. coli KRX and BL21(DE3)
	Induction of expression	Expression of T7 polymerase + IPTG, lactose
	Specific growth rate (un-/induced)	0.116 h^-1 / 0.092 h^-1
	Doubling time (un-/induced)	5.95 h / 7.51 h
Purification	Molecular weight	110.1 kDa
	Theoretical pI	5.63
	Excitation / emission	435 / 477 nm
Immobilization behaviour	Immobilization time	4 h

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 104
Illegal BglII site found at 221
Illegal XhoI site found at 1996
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 76
Illegal AgeI site found at 3913
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 493
Illegal BsaI.rc site found at 622

Expression in E. coli

The SbpA (K525401) gen was fused with a mCerulean (BBa_J18930) using [http://2011.igem.org/Team:Bielefeld-Germany/Protocols#Gibson_assembly Gibson assembly] for characterization.

The SbpA|mCerulean fusion protein was overexpressed in E. coli KRX after induction of T7 polymerase by supplementation of 0,1 % L-rhamnose and 1 mM IPTG using the [http://2011.igem.org/Team:Bielefeld-Germany/Protocols/Downstream-processing#Expression_of_S-layer_genes_in_E._coli autinduction protocol] from promega.

Figure 1: Growthcurve of E. coli KRX expressing the fusion protein of SbpA and mCerulean with and without induction. A curve depicting KRX wildtype is shown for comparsion.

Figure 2: RFU to OD₆₀₀ ratio of E. coli KRX expressing the fusion protein of SbpA and mCerulean with and without induction. A curve depicting KRX wildtype is shown for comparsion.