Difference between revisions of "Part:BBa K525223"
(→‎Expression in E. coli)
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The CspB gen was fused with a monomeric RFP ([https://parts.igem.org/Part:BBa_E1010 BBa_E1010]) using [http://2011.igem.org/Team:Bielefeld-Germany/Protocols#Gibson_assembly Gibson assembly] for characterization.
 
The CspB gen was fused with a monomeric RFP ([https://parts.igem.org/Part:BBa_E1010 BBa_E1010]) using [http://2011.igem.org/Team:Bielefeld-Germany/Protocols#Gibson_assembly Gibson assembly] for characterization.
  
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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.
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The mRFP|CspB 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.
  
 
[[Image:Bielefeld_2011_CH3_Growthcurve.png|600px|center|thumb| '''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.''']]
 
[[Image:Bielefeld_2011_CH3_Growthcurve.png|600px|center|thumb| '''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.''']]
  
 
[[Image:Bielefeld_2011_CH3_RFU_OD.png|600px|center|thumb| '''Figure 2: RFU to OD<sub>600</sub> 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.''']]
 
[[Image:Bielefeld_2011_CH3_RFU_OD.png|600px|center|thumb| '''Figure 2: RFU to OD<sub>600</sub> 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.''']]

Revision as of 22:24, 21 September 2011

S-layer cspB from Corynebacterium halotolerans with lipid anchor, 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.


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) 0.248 h-1 / 0.098 h-1
Doubling time (un-/induced) 2.79 h / 7.07 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 1102
    Illegal XhoI site found at 558
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 225
    Illegal NgoMIV site found at 1314
    Illegal NgoMIV site found at 1425
    Illegal AgeI site found at 216
    Illegal AgeI site found at 457
    Illegal AgeI site found at 504
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 906
    Illegal BsaI.rc site found at 213
    Illegal BsaI.rc site found at 591
    Illegal BsaI.rc site found at 993


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 mRFP|CspB 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.