Difference between revisions of "Part:BBa K525223"

(Identification and localisation)
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!Result
 
!Result
 
|-
 
|-
|rowspan="5"|[[Part:BBa_K525223#Expression_in_E._coli | Expression (''E. coli'')]]
+
|rowspan="4"|[[Part:BBa_K525223#Expression_in_E._coli | Expression (''E. coli'')]]
|Localisation
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|Cell membrane
+
|-
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|Compatibility
 
|Compatibility
 
|''E. coli'' KRX
 
|''E. coli'' KRX
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|Doubling time (un-/induced)
 
|Doubling time (un-/induced)
 
|2.79 h / 7.07 h
 
|2.79 h / 7.07 h
 +
|-
 +
|rowspan="8"|[[Part:BBa_K525222#Purification_of_SgsE_fusion_protein | Characterization]]
 +
|-
 +
|Number of amino acids
 +
|486
 +
|-
 +
|Molecular weight
 +
|53.4 kDa
 +
|-
 +
|Theoretical pI
 +
|4.27
 +
|-
 +
|rowspan="4"|Localization
 +
|cytoplasm
 +
|-
 +
|partially in culture supernatant
 +
|-
 +
|partially periplasm
 +
|-
 +
|not cell membrane
 
|-
 
|-
 
|}
 
|}
</center>  
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</center>
  
 
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<partinfo>BBa_K525223 parameters</partinfo>
 
<partinfo>BBa_K525223 parameters</partinfo>
 
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===Expression in ''E. coli''===
 
===Expression in ''E. coli''===
The CspB gen was fused with a monomeric RFP ([https://parts.igem.org/Part:BBa_E1010 BBa_E1010]) using [http://2011.igem.org/Team:Bielefeld-Germany/Protocols#Gibson_assembly Gibson assembly] for characterization.
+
For characterizations, the ''cspB'' gene was fused to a monomeric RFP ([https://parts.igem.org/Part:BBa_E1010 BBa_E1010]) using Gibson assembly.
  
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.
+
The CspB|mRFP fusion protein was overexpressed in ''E. coli'' KRX after induction of a T7 polymerase gene in the KRX's genome by supplementation of 0.1 % L-rhamnose using the autinduction protocol developed by Promega.
  
[[Image:Bielefeld_2011_CH3_Growthcurve.png|600px|center|thumb| '''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 OD<sub>600</sub> of the induced K525223 visibly drops when compared to the uninduced culture. While the induced culture grow significantly slower than KRX wildtype the uninduced seems to be unaffected.''']]
+
[[Image:Bielefeld_2011_CH3_Growthcurve.png|600px|center|thumb| '''Figure 1: Growth curve of ''E. coli'' KRX expressing the fusion protein of CspB and mRFP with and without induction, cultivated at 37 °C in autoinduction medium with and without inductor, respectively. A curve depicting KRX wildtype is shown for comparsion. After autoinduction at approximately 6 h the OD<sub>600</sub> of the induced <partinfo>K525222</partinfo> visibly drops when compared to the uninduced culture. While the induced culture grow significantly slower than KRX wildtype the uninduced seems to be unaffected.''']]
  
 
[[Image:Bielefeld_2011_CH3_RFU_OD.png|600px|center|thumb| '''Figure 2: RFU to OD<sub>600</sub> 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 OD<sub>600</sub> ratio starts to rise in the induced culture. Compared to the uninduced culture the ratio is roughly five times higher. Most likely due to basal transcription the RFU to OD<sub>600</sub> ratio of the uninduced culture starts to rise after 12 hours. The KRX wildtype shows no variation in the RFU to OD<sub>600</sub> ratio.''']]
 
[[Image:Bielefeld_2011_CH3_RFU_OD.png|600px|center|thumb| '''Figure 2: RFU to OD<sub>600</sub> 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 OD<sub>600</sub> ratio starts to rise in the induced culture. Compared to the uninduced culture the ratio is roughly five times higher. Most likely due to basal transcription the RFU to OD<sub>600</sub> ratio of the uninduced culture starts to rise after 12 hours. The KRX wildtype shows no variation in the RFU to OD<sub>600</sub> ratio.''']]
 +
 +
  
 
===Identification and localisation===
 
===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 ddH<sub>2</sub>O. From the other part the periplasm was detached by using a osmotic shock.  
+
After a cultivation time of 18 h the mRFP|CspB fusion protein was localized in ''E. coli'' KRX. Therefore a part of the produced biomass was mechanically disrupted and the resulting lysate was washed with ddH<sub>2</sub>O. From the other part the periplasm was detached by using an 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 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 integrates into the cell membrane with its lipid anchor. For testing this assumption the washed lysate was treated 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).
+
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.
  
In comparison with the mRFP fusion protein of [https://parts.igem.org/Part:BBa_K525224 K525224], wich has a TAT-sequence, a minor relative fluorescence in all cultivation and detergent fractions were detected (fig. 3). Together with the decreasing RFU after 9 h of cultivation (fig. 2) this results indicate 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_K525224 K525224], wich has a TAT-sequence, a minor relative fluorescence in all cultivation and detergent fractions were detected (fig. 3). Together with the decreasing RFU after 9 h of cultivation (fig. 2) this results indicate a postive effect of the TAT-sequence on the protein stability. This could be due to a digestion of <partinfo>K525223</partinfo> by proteases in the cytoplasm.  
  
[[Image:Bielefeld 2011 CH3 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 CH3 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 washed with ddH<sub>2</sub>O and resuspended 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.''']]
  
  
To obtain more specific informations about the location of the S-layer fusion protein, after comparison with same treated fraction of ''E. coli'' KRX all gel bands in a defined size area were cut out of the gel and analysed with MALDI-TOF. Results are shown in fig. 6. The fusion protein CspB/mRFP [https://parts.igem.org/Part:BBa_E1010 (BBa_E1010)] features a lipid anchor at the carboxy-terminus, but no amino-terminal TAT-sequence. In accordance with other protein variants with and without this features, the protein should be located mainly in the cytoplasm as inclusion bodies or incooperated with its lipid anchor into the cell membrane. Thus the fraction with 10 % (v/v) SDS as detergent to disintegrate the protein from the cell wall was measured with MALDI TOF. Results are shown in fig. 4.  
+
To obtain more specific information about the location of the S-layer fusion protein, after comparison with same treated fraction of ''E. coli'' KRX all gel bands in a defined size area were cut out of the gel and analysed with MALDI-TOF. Results are shown in fig. 6. The fusion protein CspB/mRFP [https://parts.igem.org/Part:BBa_E1010 (BBa_E1010)] features a lipid anchor at the carboxy-terminus, but no amino-terminal TAT-sequence. In accordance with other protein variants with and without this features, the protein should be located mainly in the cytoplasm as inclusion bodies or incooperated with its lipid anchor into the cell membrane. Thus, the fraction with 10 % (v/v) SDS as detergent to disintegrate the protein from the cell wall was measured with MALDI-TOF. Results are shown in fig. 4.  
  
  
 
[[Image:Bielefeld2011 K525233 Gel2.png|900px|thumb|right| '''Figure 4: MALDI-TOF measurement of CspB/mRFP [https://parts.igem.org/Part:BBa_E1010 (BBa_E1010)] fusion protein in different fractions. Abbreviations are Ma: Marker (PageRuler <sup>TM</sup> Prestained Protein Ladder SM0671), M (medium), PP (periplasm), L (cell lysis with ribolyser), W (wash with ddH<sub>2</sub>O). In the left half of the gel fractions of ''E. coli'' KRX with induced production of fusion protein, the right half shows fractions of ''E. coli'' KRX without carrying the plasmid coding the fusion protein. Colours show the sequence coverage of the gel lane, cutted out of the gel.''']]
 
[[Image:Bielefeld2011 K525233 Gel2.png|900px|thumb|right| '''Figure 4: MALDI-TOF measurement of CspB/mRFP [https://parts.igem.org/Part:BBa_E1010 (BBa_E1010)] fusion protein in different fractions. Abbreviations are Ma: Marker (PageRuler <sup>TM</sup> Prestained Protein Ladder SM0671), M (medium), PP (periplasm), L (cell lysis with ribolyser), W (wash with ddH<sub>2</sub>O). In the left half of the gel fractions of ''E. coli'' KRX with induced production of fusion protein, the right half shows fractions of ''E. coli'' KRX without carrying the plasmid coding the fusion protein. Colours show the sequence coverage of the gel lane, cutted out of the gel.''']]
  
Fig. 4 shows, that the protein could be identified in all measured gel bands. The results indicate, that the protein is incooperated into the cell membrane. No fluoresence could be detected in the fractions using urea as detergent (see fig. 3), thus the protein probably does not form inclusion bodies.
+
Fig. 4 shows, that the protein could be identified in all measured gel bands. The results indicate, that the protein is incorperated into the cell membrane. No fluoresence could be detected in the fractions using urea as detergent (see fig. 3), thus the protein probably does not form inclusion bodies.  
  
 
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Revision as of 02:07, 22 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) 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
Characterization
Number of amino acids 486
Molecular weight 53.4 kDa
Theoretical pI 4.27
Localization cytoplasm
partially in culture supernatant
partially periplasm
not cell membrane

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

For characterizations, the cspB gene was fused to a monomeric RFP (BBa_E1010) using Gibson assembly.

The CspB|mRFP fusion protein was overexpressed in E. coli KRX after induction of a T7 polymerase gene in the KRX's genome by supplementation of 0.1 % L-rhamnose using the autinduction protocol developed by Promega.

Figure 1: Growth curve of E. coli KRX expressing the fusion protein of CspB and mRFP with and without induction, cultivated at 37 °C in autoinduction medium with and without inductor, respectively. A curve depicting KRX wildtype is shown for comparsion. After autoinduction at approximately 6 h the OD600 of the induced BBa_K525222 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 five times higher. 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 was localized in E. coli KRX. Therefore a part of the produced biomass was mechanically disrupted and the resulting lysate was washed with ddH2O. From the other part the periplasm was detached by using an 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 integrates into the cell membrane with its lipid anchor. For testing this assumption the washed lysate was treated 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 K525224, wich has a TAT-sequence, a minor relative fluorescence in all cultivation and detergent fractions were detected (fig. 3). Together with the decreasing RFU after 9 h of cultivation (fig. 2) this results indicate a postive effect of the TAT-sequence on the protein stability. This could be due to a digestion of BBa_K525223 by proteases in the cytoplasm.

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 washed with ddH2O and resuspended 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.


To obtain more specific information about the location of the S-layer fusion protein, after comparison with same treated fraction of E. coli KRX all gel bands in a defined size area were cut out of the gel and analysed with MALDI-TOF. Results are shown in fig. 6. The fusion protein CspB/mRFP (BBa_E1010) features a lipid anchor at the carboxy-terminus, but no amino-terminal TAT-sequence. In accordance with other protein variants with and without this features, the protein should be located mainly in the cytoplasm as inclusion bodies or incooperated with its lipid anchor into the cell membrane. Thus, the fraction with 10 % (v/v) SDS as detergent to disintegrate the protein from the cell wall was measured with MALDI-TOF. Results are shown in fig. 4.


Figure 4: MALDI-TOF measurement of CspB/mRFP (BBa_E1010) fusion protein in different fractions. Abbreviations are Ma: Marker (PageRuler TM Prestained Protein Ladder SM0671), M (medium), PP (periplasm), L (cell lysis with ribolyser), W (wash with ddH2O). In the left half of the gel fractions of E. coli KRX with induced production of fusion protein, the right half shows fractions of E. coli KRX without carrying the plasmid coding the fusion protein. Colours show the sequence coverage of the gel lane, cutted out of the gel.

Fig. 4 shows, that the protein could be identified in all measured gel bands. The results indicate, that the protein is incorperated into the cell membrane. No fluoresence could be detected in the fractions using urea as detergent (see fig. 3), thus the protein probably does not form inclusion bodies.