Difference between revisions of "Part:BBa K525224"

(Identification and localisation)
(Identification and localisation)
Line 69: Line 69:
 
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 mRFP|CspB out of the membranes, if it intigrates. From the other part of the cells the periplasm was detached by using a osmotic shock. The existance of fluorescene in the periplasm fraction, showed in fig. 3, indicates that ''C. halotolerans'' TAT-signal sequence is at least in part functional in E. coli KRX.
 
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 mRFP|CspB out of the membranes, if it intigrates. From the other part of the cells the periplasm was detached by using a osmotic shock. The existance of fluorescene in the periplasm fraction, showed in fig. 3, indicates that ''C. halotolerans'' TAT-signal sequence is at least in part functional in E. coli KRX.
  
Specific for K525224 fused with mRFP is the proportional to the K525222 and K525223 fusion proteins high fluorescence in the culture supernatant. This indicates that the fusion protein is secreted into the periplasm via the TAT-pathway and partly relesed into the culture medium. Because there is no known release pathway for S-layer proteins in ''E. coli'' the periplasm might burst in consequence of the overexpression.  
+
Specific for K525224 fused with mRFP is the proportional to the mRFP fusion proteins of K525222 and K525223 high fluorescence in the culture supernatant. This indicates that the fusion protein is secreted into the periplasm via the TAT-pathway and partly relesed into the culture medium. Because there is no known release pathway for S-layer proteins in ''E. coli'' the periplasm might burst in consequence of the overexpression.  
  
The fluorescence in all cultivation fractions plus the fluorescence in the lysis und wash fraction shows that the fusion protein is water soluble and doesn't sediment doesn't during centrifugation. Together with the flourescence in 4 of 5 of the detergent fractions indicate that a part of the produced proteins form inclusion bodies.
+
The fluorescence in all cultivation fractions plus the fluorescence in the lysis und wash fraction shows that the fusion protein is water soluble and doesn't sediment during centrifugation. The absence of fluotrescence indicates that the expressed fusion protein doesn't for inclusion bodies.
  
  
 
[[Image:Bielefeld 2011 CH4 Purification.png|700px|thumb|center| '''Figure 3: Fluorescence progression of the mRFP[https://parts.igem.org/Part:BBa_E1010 (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 ddH<sub>2</sub>O 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.''']]
 
[[Image:Bielefeld 2011 CH4 Purification.png|700px|thumb|center| '''Figure 3: Fluorescence progression of the mRFP[https://parts.igem.org/Part:BBa_E1010 (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 ddH<sub>2</sub>O 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.''']]
 
<!-- -->
 
<!-- -->

Revision as of 01:44, 22 September 2011

S-layer cspB from Corynebacterium halotolerans with TAT-sequence, 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) 0.251 h-1 / 0.157 h-1
Doubling time (un-/induced) 2.76 h / 4.42 h

After processing:

Molecular weight: 50.6 kDa

Theoretical pI: 4.25


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 440
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 440
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1191
    Illegal XhoI site found at 647
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 440
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 440
    Illegal NgoMIV site found at 314
    Illegal NgoMIV site found at 1403
    Illegal AgeI site found at 89
    Illegal AgeI site found at 305
    Illegal AgeI site found at 546
    Illegal AgeI site found at 593
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 995
    Illegal BsaI.rc site found at 302
    Illegal BsaI.rc site found at 680
    Illegal BsaI.rc site found at 1082


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, cultivated at 37 °C in autoinduction medium with, respectively, without inductor. A curve depicting KRX wildtype is shown for comparsion. After induction at approximately 6 h the OD600 of the induced K525224 visibly drops when compared to the uninduced culture. While the induced culture grow significantly slower than KRX wildtype the uninduced seems to be unaffected.
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. After induction at approximately 6 h the RFU to OD600 ratio starts to rise in the induced culture. Compared to the uninduced culture the ratio is roughly seven times higher at its highest point but starts to drop during the cultivation due to degradation of the fusion protein. Most likely due to basal transcription the RFU to OD600 ratio of the uninduced culture starts to rise after 12 hours. The KRX wildtype shows no variation in the RFU to OD600 ratio.

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 mRFP|CspB out of the membranes, if it intigrates. From the other part of the cells the periplasm was detached by using a osmotic shock. The existance of fluorescene in the periplasm fraction, showed in fig. 3, indicates that C. halotolerans TAT-signal sequence is at least in part functional in E. coli KRX.

Specific for K525224 fused with mRFP is the proportional to the mRFP fusion proteins of K525222 and K525223 high fluorescence in the culture supernatant. This indicates that the fusion protein is secreted into the periplasm via the TAT-pathway and partly relesed into the culture medium. Because there is no known release pathway for S-layer proteins in E. coli the periplasm might burst in consequence of the overexpression.

The fluorescence in all cultivation fractions plus the fluorescence in the lysis und wash fraction shows that the fusion protein is water soluble and doesn't sediment during centrifugation. The absence of fluotrescence indicates that the expressed fusion protein doesn't for inclusion bodies.


Figure 3: Fluorescence 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.