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	136.9 kDa
	Theoretical pI	4.89
	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 gene under the control of a T7 / lac promoter (BBa_K525403) was fused to mCerulean (BBa_J18930) using Freiburg BioBrick assembly for characterization experiments.

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 autinduction protocol by Promega.

Figure 1: Growth curve of E. coli KRX expressing the fusion protein of SbpA and mCerulean with and without induction, cultivated at 37 °C in autoinduction medium with and without inductor, respectively. A curve depicting KRX wildtype is shown for comparsion. After induction at approximately 6 h the OD₆₀₀ of the induced K525406 visibly drops when compared to the uninduced culture. Both cultures grow significantly slower than KRX wildtype.

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. After induction at approximately 6 h the RFU to OD₆₀₀ ratio starts to rise in the induced culture. Compared to the uninduced culture the ratio is roughly three times higher. The KRX wildtype shows no variation in the RFU to OD₆₀₀ ratio.

Methods

Expression of S-layer genes in E. coli

Chassis: Promega's [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ E. coli KRX]

Medium: LB medium supplemented with 20 mg L^-1 chloramphenicol
- For autoinduction: Cultivations in LB-medium were supplemented with 0.1 % L-rhamnose and 1 mM IPTG as inducer and 0.05 % glucose

Measuring of mCerulean

Take at least 500 µL sample for each measurement (200 µL is needed for one measurement) so you can perform a repeat determination
Freeze biological samples at -80 °C for storage, keep cell-free at 4 °C in the dark
To measure the samples thaw at room temperature and fill 200 µL of each sample in one well of a black, flat bottom 96 well microtiter plate (perform at least a repeat determination)
Measure the fluorescence in a platereader (we used a [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1812&ID=1916&Menu=1&Item=21.2.10.1 Tecan Infinite® M200 platereader]) with following settings:
- 20 sec orbital shaking (1 mm amplitude with a frequency of 87.6 rpm)
- Measurement mode: Top
- Excitation: 433 nm
- Emission: 475 nm
- Number of reads: 25
- Manual gain: 100
- Integration time: 20 µs

References

Kainz B, Steiner K, Möller M, Pum D, Schäffer C, Sleytr UB, Toca-Herrera JL (2010) Absorption, Steady-State Fluorescence, Fluorescence Lifetime, and 2D Self-Assembly Properties of Engineered Fluorescent S-Layer Fusion Proteins of Geobacillus stearothermophilus NRS 2004/3a, [http://pubs.acs.org/doi/abs/10.1021/bm901071b Biomacromolecules 11(1):207-214].

Sleytr UB, Huber C, Ilk N, Pum D, Schuster B, Egelseer EM (2007) S-layers as a tool kit for nanobiotechnological applications, [http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2006.00573.x/full FEMS Microbiol Lett 267(2):131-144].