Difference between revisions of "Part:BBa K525222"

(Identification and localisation)
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===Identification and localisation===
 
===Identification and localisation===
After a cultivation time of 18 h the CspB|mRFP 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 ddH<sub>2</sub>O. From the other part the periplasm was detached by using a osmotic shock. The existance of fluorescene in the periplasma fraction, showed in fig. 3, indicates that ''Brevibacterium flavum'' TAT-signal sequence is at least in part functional in ''E. coli'' KRX.
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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 ddH<sub>2</sub>O. Then the lysate was treted with ionic, nonionic and zwitterionic detergents to release the CspB|mRFP out of the membranes, if it intigrates. From the other part the periplasm was detached by using a osmotic shock.
  
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 CspB|mRFP out of the membranes.
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The fluorescence in all cultivation fractions plus the fluorescence in the lysis und wash fraction shows that the fusion protein is water soluble and sediment not during centrifugation. Together with the absence of flourescence in the detergent fractions verifies that the fusion protein is not integrated into the cell membrane (fig. 3) and is not present as inclusion body.
 
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The existance of flourescence in the detergent fractions and the non-existancing 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).
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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. The MALDI-TOF analysis of the relevant size range in the polyacrylamid gel approved the existance of the intact fusion protein in all detergent fractions (fig x).
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In comparison with the mRFP fusion protein of [https://parts.igem.org/Part:BBa_K525121 K525121], wich has a lipid anchor, a minor relative fluorescence per OD<sub>600</sub> in all cultivation and detergent fractions was detected (fig. 3). Together with the decreasing RFU/OD<sub>600</sub> after 12 h of cultivation (fig. 2) indicates this rusult a postive effect of the lipid anchor on the protein stability.
 
In comparison with the mRFP fusion protein of [https://parts.igem.org/Part:BBa_K525121 K525121], wich has a lipid anchor, a minor relative fluorescence per OD<sub>600</sub> in all cultivation and detergent fractions was detected (fig. 3). Together with the decreasing RFU/OD<sub>600</sub> after 12 h of cultivation (fig. 2) indicates this rusult a postive effect of the lipid anchor on the protein stability.

Revision as of 22:13, 21 September 2011

S-layer cspB from Corynebacterium halotolerans

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) 0.245 h-1 / 0.109 h-1
Doubling time (un-/induced) 2.83 h / 6.33 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 1103
    Illegal XhoI site found at 559
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 226
    Illegal NgoMIV site found at 1315
    Illegal AgeI site found at 217
    Illegal AgeI site found at 458
    Illegal AgeI site found at 505
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 907
    Illegal BsaI.rc site found at 214
    Illegal BsaI.rc site found at 592
    Illegal BsaI.rc site found at 994


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 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.

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. Then the lysate was treted with ionic, nonionic and zwitterionic detergents to release the CspB|mRFP out of the membranes, if it intigrates. From the other part the periplasm was detached by using a osmotic shock.

The fluorescence in all cultivation fractions plus the fluorescence in the lysis und wash fraction shows that the fusion protein is water soluble and sediment not during centrifugation. Together with the absence of flourescence in the detergent fractions verifies that the fusion protein is not integrated into the cell membrane (fig. 3) and is not present as inclusion body.

In comparison with the mRFP fusion protein of K525121, wich has a lipid anchor, a minor relative fluorescence per OD600 in all cultivation and detergent fractions was detected (fig. 3). Together with the decreasing RFU/OD600 after 12 h of cultivation (fig. 2) indicates this rusult a postive effect of the lipid anchor on the protein stability.

Figure 3: Fluorescence pro OD600 progression of the CspB/mRFP (BBa_E1010) 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.