Part:BBa_K525224

S-layer cspB from Corynebacterium halotolerans with TAT-sequence, PT7 and RBS 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.

Important parameters

Experiment	Characteristic	Result
Expression (E. coli)	Localisation	Cell membrane
	Compatibility	E. coli KRX
	Induction of expression	L-rhamnose for induction of T7 polymerase
	Specific growth rate (un-/induced)	0.251 h-1 / 0.157 h-1
	Doubling time (un-/induced)	2.76 h / 4.42 h

After processing:

Molecular weight: 50.6 kDa

Theoretical pI: 4.25

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 440
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 440
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1191
  Illegal XhoI site found at 647
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 440
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 440
  Illegal NgoMIV site found at 314
  Illegal NgoMIV site found at 1403
  Illegal AgeI site found at 89
  Illegal AgeI site found at 305
  Illegal AgeI site found at 546
  Illegal AgeI site found at 593
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 995
  Illegal BsaI.rc site found at 302
  Illegal BsaI.rc site found at 680
  Illegal BsaI.rc site found at 1082
  

Expression in E. coli

The CspB gen was fused with a monomeric RFP (BBa_E1010) using [http://2011.igem.org/Team:Bielefeld-Germany/Protocols#Gibson_assembly Gibson assembly] for characterization.

The CspB|mRFP fusion protein was overexpressed in E. coli KRX after induction of T7 polymerase by supplementation of 0,1 % L-rhamnose 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 CspB and mRFP 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 CspB and mRFP with and without induction. A curve depicting KRX wildtype is shown for comparsion.