Difference between revisions of "Part:BBa K525406"

Revision as of 23:10, 21 September 2011

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 (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, cultivated at 37 °C in autoinduction medium with, respectively, without inductor. 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.

@@ Line 73: / Line 73: @@
 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.
-[[Image:Bielefeld_2011_406_Growthcurve.png|600px|center|thumb| '''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.''']]
+[[Image:Bielefeld_2011_406_Growthcurve.png|600px|center|thumb| '''Figure 1: Growthcurve of ''E. coli'' KRX expressing the fusion protein of SbpA and mCerulean with and without induction, cultivated at 37 °C in autoinduction medium with, respectively, without inductor. A curve depicting KRX wildtype is shown for comparsion. After induction at approximately 6 h the OD<sub>600</sub> of the induced K525406 visibly drops when compared to the uninduced culture. Both cultures grow significantly slower than KRX wildtype.''']]
-[[Image:Bielefeld_2011_406_RFU_OD.png|600px|center|thumb| '''Figure 2: RFU to OD<sub>600</sub> 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.''']]
+[[Image:Bielefeld_2011_406_RFU_OD.png|600px|center|thumb| '''Figure 2: RFU to OD<sub>600</sub> 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 OD<sub>600</sub> 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 OD<sub>600</sub> ratio.''']]