Difference between revisions of "Part:BBa K525304"

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The SgsE|mCherry RFP 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|mCherry RFP 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_304_Growthcurve1.png|600px|center|thumb| '''Figure 1: Growthcurve of ''E. coli'' KRX expressing the fusion protein of Sgse and mCherry RFP with and without induction. A curve depicting KRX wildtype is shown for comparsion.''']]
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[[Image:Bielefeld_2011_304_Growthcurve1.png|600px|center|thumb| '''Figure 1: Growthcurve of ''E. coli'' KRX expressing the fusion protein of Sgse and mCherry RFP with and without induction. A curve depicting KRX wildtype is shown for comparsion. After induction at approximately 6 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.''']]
  
 
[[Image:Bielefeld_2011_304_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 mCherry RFP with and without induction. A curve depicting KRX wildtype is shown for comparsion.''']]
 
[[Image:Bielefeld_2011_304_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 mCherry RFP with and without induction. A curve depicting KRX wildtype is shown for comparsion.''']]

Revision as of 21:23, 21 September 2011

Fusion Protein of S-Layer SgsE and mCherry RFP

Fusion protein of S-layer SgsE and mCherry RFP

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 mCherry as a pH indicator ([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) 0.128 h-1 / 0.094 h-1
Doubling time (un-/induced) 5.41 h / 7.39 h
Purification Molecular weight 109.9 kDa
Theoretical pI 5.70
Excitation / emission 587 / 610 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 3112
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1657


Expression in E. coli

The SgsE (K525301) gen was fused with a mCherry RFP (BBa_J18932) using [http://2011.igem.org/Team:Bielefeld-Germany/Protocols#Gibson_assembly Gibson assembly] for characterization.

The SgsE|mCherry RFP 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 mCherry RFP with and without induction. A curve depicting KRX wildtype is shown for comparsion. After induction at approximately 6 h the OD600 of the induced K525304 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 mCherry RFP with and without induction. A curve depicting KRX wildtype is shown for comparsion.