Difference between revisions of "Part:BBa K525306"

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The SgsE|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.
 
The SgsE|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.
  
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[[Image:Bielefeld_2011_306_Growthcurve.png|600px|center|thumb| '''Figure 1: Growthcurve of ''E. coli'' KRX expressing the fusion protein of Sgse and mCerluean 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 4 h the OD<sub>600</sub> of the induced K525304 visibly drops when compared to the uninduced culture. Both cultures grow significantly slower than KRX wildtype.''']]
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[[Image:Bielefeld_2011_306_Growthcurve.png|600px|center|thumb| '''Figure 1: Growthcurve of ''E. coli'' KRX expressing the fusion protein of Sgse and mCerluean 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 4 h the OD<sub>600</sub> of the induced K525306 visibly drops when compared to the uninduced culture. Both cultures grow significantly slower than KRX wildtype.''']]
  
 
[[Image:Bielefeld_2011_306_RFU_OD.png|600px|center|thumb| '''Figure 2: RFU to OD<sub>600</sub> ratio of ''E. coli'' KRX expressing the fusion protein of Sgse and mCerulean with and without induction. A curve depicting KRX wildtype is shown for comparsion. After induction at approximately 4 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 34 times higher at its highest point but starts to drop during the cultivation due to degradation of the fusion protein. The KRX wildtype shows no variation in the RFU to OD<sub>600</sub> ratio.''']]
 
[[Image:Bielefeld_2011_306_RFU_OD.png|600px|center|thumb| '''Figure 2: RFU to OD<sub>600</sub> ratio of ''E. coli'' KRX expressing the fusion protein of Sgse and mCerulean with and without induction. A curve depicting KRX wildtype is shown for comparsion. After induction at approximately 4 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 34 times higher at its highest point but starts to drop during the cultivation due to degradation of the fusion protein. The KRX wildtype shows no variation in the RFU to OD<sub>600</sub> ratio.''']]

Revision as of 23:03, 21 September 2011

Fusion Protein of S-Layer SgsE and mCerulean

Bielefeld-Germany2011-S-Layer-Geometrien.jpg

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 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 or lactose
Specific growth rate (un-/induced) 0.127 h-1 / 0.229 h-1
Doubling time (un-/induced) 5.45 h / 3.02 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 167
    Illegal BglII site found at 1022
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 76
    Illegal AgeI site found at 3121
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1657


Expression in E. coli

The SgsE (K525301) gen was fused with a mCerulean (BBa_J18930) using [http://2011.igem.org/Team:Bielefeld-Germany/Protocols#Gibson_assembly Gibson assembly] for characterization.

The SgsE|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 Sgse and mCerluean 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 4 h the OD600 of the induced K525306 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 Sgse and mCerulean with and without induction. A curve depicting KRX wildtype is shown for comparsion. After induction at approximately 4 h the RFU to OD600 ratio starts to rise in the induced culture. Compared to the uninduced culture the ratio is roughly 34 times higher at its highest point but starts to drop during the cultivation due to degradation of the fusion protein. The KRX wildtype shows no variation in the RFU to OD600 ratio.