Difference between revisions of "Part:BBa K525121"

(Identification and localisation)
(Identification and localisation)
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[[Image:Bielefeld 2011 BF1 Purification.png|700px|thumb|center| '''Figure 3: Fluorescence pro OD<sub>600</sub> progression of the CspB/mRFP [https://parts.igem.org/Part:BBa_E1010 (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 ddH<sub>2</sub>O and resuspendet in the respective detergent. The used detergent acronyms stand for: SDS = 10 % (v/v) sodium dodecyl sulfate; UTU = 7 M urea and 3 M thiourea; U = 10 M urea; NLS = 10 % (v/v) n-lauroyl sarcosine; CHAPS = 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.''']]
 
[[Image:Bielefeld 2011 BF1 Purification.png|700px|thumb|center| '''Figure 3: Fluorescence pro OD<sub>600</sub> progression of the CspB/mRFP [https://parts.igem.org/Part:BBa_E1010 (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 ddH<sub>2</sub>O and resuspendet in the respective detergent. The used detergent acronyms stand for: SDS = 10 % (v/v) sodium dodecyl sulfate; UTU = 7 M urea and 3 M thiourea; U = 10 M urea; NLS = 10 % (v/v) n-lauroyl sarcosine; CHAPS = 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.''']]
  
MALDI TOF analysis first used to identify the location of the fusion protein in different fractions. After centrifugation the supernatant of the media was collected, a periplasmatic fraction was achieved after osmotic shock [http://2011.igem.org/Team:Bielefeld-Germany/Protocols/Downstream-processing#Release_of_periplasmic_protein_fraction_from_E._coli_by_cold_osmotic_shock see protocol]. Complete cell lysis was achieved
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MALDI TOF analysis first used to identify the location of the fusion protein in different fractions. Fractions of media supernatant after cultivation, periplasmatic
 
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Revision as of 19:43, 21 September 2011

S-layer cspB from Corynebacterium glutamicum with TAT-Sequence and lipid anchor, PT7 and RBS


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.

This fluorescent S-layer fusion protein is used to characterize purification methods and the S-layer's ability to self-assemble on surfaces.

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
Characteristics Molecular weight
Theoretical pI

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 1421
    Illegal XhoI site found at 248
    Illegal XhoI site found at 866
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1400
    Illegal SapI site found at 647
    Illegal SapI site found at 859
    Illegal SapI site found at 1407

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. Cultivations were carried out in autoinduction medium at 37 ˚C.
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. Cultivations were carried out in autoinduction medium at 37 ˚C.

Identification and localisation

After a cultivation time of 18 h the CspB|mRFP fusion protein has to be localized in E. coli KRX. Therefore a part of the produced biomass was mechanically disrupted and the resulting lysate was washed with ddH2O. The periplasm was detached by using a osmotic shock from another part of the cells. The existance of fluorescene in the periplasm 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 and the cell depris were still red. 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.

The existance of flourescence in the detergent fractions and the proportionally low fluorescence in the wash fraction confirm the hypothesis 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).

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 % (v/v) sodium dodecyl sulfate; UTU = 7 M urea and 3 M thiourea; U = 10 M urea; NLS = 10 % (v/v) n-lauroyl sarcosine; CHAPS = 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.

MALDI TOF analysis first used to identify the location of the fusion protein in different fractions. Fractions of media supernatant after cultivation, periplasmatic