Difference between revisions of "Part:BBa K525223"

(Identification and localisation)
(Identification and localisation)
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The S-layer fusion protein could not be found in the polyacrylamide gel after a SDS-PAGE of the lysate. This indicated that the fusion protein intigrates into the cell membrane with its lipid  anchor. For testing this  assumption the washed lysate was treted with ionic, nonionic and zwitterionic detergents to release the mRFP|CspB out of the membranes.
 
The S-layer fusion protein could not be found in the polyacrylamide gel after a SDS-PAGE of the lysate. This indicated that the fusion protein intigrates into the cell membrane with its lipid  anchor. For testing this  assumption the washed lysate was treted with ionic, nonionic and zwitterionic detergents to release the mRFP|CspB out of the membranes.
  
The existance of flourescence in two of the detergent fractions (10 % SDS and 10 % SDS) and the low fluorescence in the wash fraction confirm the hypothesis of an insertion into the cell membrane (fig. 3). An insertion of these S-layer proteins might stabilize the membrane structure and increase the stability of cells against mechanical and chemical treatment. A stabilization of ''E. coli'' expressing S-lyer proteins was discribed by Lederer ''et al.'', (2010).
+
The existance of flourescence in two of the detergent fractions (10 % SDS and 10 % n-lauroyl sarcosine) and the low fluorescence in the wash fraction confirm the hypothesis of an insertion into the cell membrane (fig. 3). An insertion of these S-layer proteins might stabilize the membrane structure and increase the stability of cells against mechanical and chemical treatment. A stabilization of ''E. coli'' expressing S-lyer proteins was discribed by Lederer ''et al.'', (2010).
  
 
An other important fact is, that there is actually mRFP fluorescence measurable in such high concentrated detergent solutions. The S-layer seems to stabilize the biologically active conformation of mRFP.
 
An other important fact is, that there is actually mRFP fluorescence measurable in such high concentrated detergent solutions. The S-layer seems to stabilize the biologically active conformation of mRFP.

Revision as of 23:04, 21 September 2011

S-layer cspB from Corynebacterium halotolerans with lipid anchor, PT7 and RBS

Bielefeld-Germany2011-S-Layer-Geometrien.jpg

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.

Identification and localisation

After a cultivation time of 18 h the mRFP|CspB fusion protein has to be localized in E. coli KRX. Therefor a part of the produced biomass was mechanically disrupted and the resulting lysate was wahed with ddH2O. From the other part the periplasm was detached by using a osmotic shock. The high of fluorescene in the periplasma fraction, showed in fig. 3, indicates that Corynebacterium halotolerans TAT-signal sequence is at least in part functional in E. coli KRX.

The S-layer fusion protein could not be found in the polyacrylamide gel after a SDS-PAGE of the lysate. This indicated that the fusion protein intigrates into the cell membrane with its lipid anchor. For testing this assumption the washed lysate was treted with ionic, nonionic and zwitterionic detergents to release the mRFP|CspB out of the membranes.

The existance of flourescence in two of the detergent fractions (10 % SDS and 10 % n-lauroyl sarcosine) and the low fluorescence in the wash fraction confirm the hypothesis of an insertion into the cell membrane (fig. 3). An insertion of these S-layer proteins might stabilize the membrane structure and increase the stability of cells against mechanical and chemical treatment. A stabilization of E. coli expressing S-lyer proteins was discribed by Lederer et al., (2010).

An other important fact is, that there is actually mRFP fluorescence measurable in such high concentrated detergent solutions. The S-layer seems to stabilize the biologically active conformation of mRFP.

In comparison with the mRFP fusion protein of ???, wich has a TAT-sequence, a minor relative fluorescence per OD600 in all cultivation and detergent fractions was detected (fig. 3). Together with the decreasing RFU/OD600 after 9 h of cultivation (fig. 2) this results indicate a postive effect of the TAT-sequence on the protein stability.

Figure 3: Fluorescence pro OD600 progression of the mRFP(BBa_E1010)/CspB fusion protein initiating with the cultivation fractions up to the detergent fractions of the seperate denaturations. Cultivations were carried out in autoinduction medium at 37 ˚C. The cells were mechanically disrupted and the resulting biomass was wahed with ddH2O and resuspendet in the respective detergent. The used detergent acronyms stand for: SDS = 10 % sodium dodecyl sulfate; UTU = 7 M urea and 3 M thiourea; U = 10 M urea; NLS = 10 % n-lauroyl sarcosine; 2 % CHAPS = 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.