Difference between revisions of "Part:BBa K525123"

(→‎Identification and localisation)
(→‎Identification and localisation)
Line 66: Line 66:
 
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).
 
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).
  
−
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).
+
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 [https://parts.igem.org/Part:BBa_K525121 K525121], wich has a TAT-sequence, 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 rusults in a postive effect of the TAT-sequence on the protein stability.
 
In comparison with the mRFP fusion protein of [https://parts.igem.org/Part:BBa_K525121 K525121], wich has a TAT-sequence, 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 rusults in a postive effect of the TAT-sequence on the protein stability.

Revision as of 22:36, 21 September 2011

S-layer cspB from Corynebacterium glutamicum with lipid anchor and 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.

The S-layer of C. glutamicum is characterized by a hexagonal lattice symmetry. Attachment between S-layer and cell wall was found to be due to the hydrophobic carboxy-terminus of the PS2 protein [http://www.sciencedirect.com/science/article/pii/S016816560400241X Hansmeier et al., 2004]).

Important parameters

Experiment Characteristic Result
Expression (E. coli) Localisation cell membrane
Compatibility E. coli KRX
Inductor for expression L-rhamnose for induction of T7 polymerase
Specific growth rate (un-/induced) 0.245 h-1 / 0.093 h-1
Doubling time (un-/induced) 2.82 h / 7.42 h

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1334
    Illegal XhoI site found at 161
    Illegal XhoI site found at 779
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1313
    Illegal SapI site found at 560
    Illegal SapI site found at 772
    Illegal SapI site found at 1320


Expression in E. coli

For characterization the CspB gen was fused with a monomeric RFP (BBa_E1010) using Gibson assembly.

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 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 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.

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 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).

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 K525121, 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 12 h of cultivation (fig. 2) indicates this rusults in 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.