Team:Bielefeld-Germany/Results/S-Layer

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(Summary of results)
(The S-layer protein PS2 of Corynbacterium halotolerans)
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==The S-layer protein PS2 of ''Corynbacterium halotolerans''==
==The S-layer protein PS2 of ''Corynbacterium halotolerans''==
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* Characterization of expression and induction of different variants of CspB from ''Corynebacterium halotolerans'' fused with a monomeric RFP (([http://partsregistry.org/Part:BBa_E1010 BBa_E1010]). Expression was observed through measurement of optical density and fluorescence. Click on the construct to show the results.
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* Characterization of expression and induction of different variants of CspB from ''Corynebacterium halotolerans'' fused with a monomeric RFP ([http://partsregistry.org/Part:BBa_E1010 BBa_E1010]). Expression was observed through measurement of optical density and fluorescence. Click on the construct to show the results.
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Revision as of 13:14, 28 October 2011


Contents

Summary of results

Four different S-layer BioBricks with different lattice structures were created and sent to the partsregistry. The behaviour of these genes when expressed in E. coli were characterized and purification strategies for the expressed proteins were developed. Two purified fluorescent S-layer fusion proteins from different organisms were immobilized on beads, leading to a highly significant fluorescence enhancement of these beads (p < 10-14). Furthermore regarding the other two S-layers (CspB from Corynebacterium glutamicum and Corynebacterium halotolerans) we discovered that while expression with a lipid anchor resulted in an integration into the cell membrane, the expression with a TAT-sequence resulted in a segregation into the medium. We also detected, that those S-layers seem to stabilize the biologically active conformation of mRFP. Furthermore we expressed and purified a fluorescent CspB fusion protein from C. halotolerans which has never been expressed in E. coli until now.





The S-layer protein PS2 of Corynbacterium glutamicum

  • Characterization of expression and induction of different variants of CspB fused with a monomeric RFP (BBa_E1010). Expression was observed through measurement of optical density and fluorescence. Click on the construct to show the results.
Biobrick Number Construct
K525121  CspB with TAT-sequence and lipid anchor 
K525123  CspB without TAT-sequence and with lipid anchor 


  • Measurement of fluoresence to identify the location of the fusion protein in different fractions of the cells. Fractions were washed cells, the periplasm fraction after disruption of the periplasm, the supernatant of the medium after cultivation, the wash fraction of the pellet after lysis and wash with ddH2O, as well as the fractions after treating the pellet from lysis with different detergents.
  • Identification of location of fusion protein in the cell through fractionation with different detergents and periplasmatic disruption. Identification of protein-containing fractions with fluorescence measurement and MALDI-TOF. Table below shows the supposed location of the fusion protein in the cell, depending on presence of lipid anchor and TAT-sequence. Click on the construct to show the results.
 Biobrick Number  Construct  Supernatant   Periplasm   Cell lysis   Integration in cell membrane   Inclusion bodies 
K525121  CspB with TAT-sequence and lipid anchor       X                     X
K525123  CspB without TAT-sequence and with lipid anchor         X                     X


Complete Results

The S-layer protein PS2 of Corynbacterium halotolerans

  • Characterization of expression and induction of different variants of CspB from Corynebacterium halotolerans fused with a monomeric RFP (BBa_E1010). Expression was observed through measurement of optical density and fluorescence. Click on the construct to show the results.
Biobrick Number Construct
K525222  CspB without TAT-sequence and lipid anchor
K525223  CspB without TAT-sequence and with lipid anchor
K525224  CspB with TAT-sequence and without lipid anchor


  • Measurement of fluoresence to identify the location of the fusion protein in different fractions of the cells. Fractions were washed cells, the periplasm fraction after disruption of the periplasm, the supernatant of the medium after cultivation, the wash fraction of the pellet after lysis and wash with ddH2O, as well as the fractions after treating the pellet from lysis with different detergents.
  • Identification of location of fusion protein in the cell through fractionation with different detergents and periplasmatic disruption. Identification of protein-containing fractions with fluorescence measurement and MALDI-TOF. Table below shows the supposed location of the fusion protein in the cell, depending on presence of lipid anchor and TAT-sequence. Small x show small amounts of protein, large X show the main amount of protein. Click on the construct to show the results.
 Biobrick Number  Construct  Supernatant   Periplasm   Cell lysis   Integration in cell membrane   Inclusion bodies
K525222  CspB without TAT-sequence and lipid anchor            X         x                        x
K525223  CspB without TAT-sequence and with lipid anchor            x         x
K525224  CspB with TAT-sequence and without lipid anchor            X         x       x


  • Purification of the protein could be achieved through precipitation with ammonium sulfate, followed by ultra- and diafiltration. Salt concentration in the final anion exchange chromatography could be optimized to 400 mM NaCl (See: Purification and final strategy).


Complete Results

The S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a

  • Characterization of expression and induction of K525305, a translational fusion of the sgsE gene (K525303) with mCitrine (J18931). Expression was observed through measurement of fluorescence intensity and optical density (See: Expression in E. coli).
  • Isolation and purification of the inclusion bodies using detergents, ultra- and diafiltration. Providing of water-soluble fusion protein monomers for recrsytallisation and coating through dialyzation (See: Purification of SgsE fusion protein inclusion bodies).
  • Characterization of immobilization behaviour of proteins coated to silica beads through measurement of fluorescence of supernatant, the wash fraction and the beads (See: Immobilization behaviour).
  • Immobilization kinetics to determine the optimal bead to protein ratio (See: bead to protein ratio). Data could be fitted to a dose-response function. A good silica bead conentration for immobilization of 100 µg protein could be calculated to be around 150 - 200 mg mL-1.


Complete Results

The S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177

  • Characterization of the expression of K525405, a fusion of the sgsE gene (K525403) with mCitrine (J18931). Expression was observed through measurement of fluorescence intensity and optical density (See: Expression in E. coli).
  • Isolation and purifciation of the inclusion bodies using different detergents, ultra- and diafiltration. Providing of water-soluble fusion protein monomers for recrystallization and coating experiments through dialysis (See: Purification of SbpA fusion protein inclusion bodies).
  • Immobilization kinetics to determine optimal bead to protein ratio (See: Immobilization behaviour). Data could be fitted to a dose-response function. A good silica bead concentration for immobilization of 100 µg protein could be calculated to be around 200 - 250 mg mL-1.


Complete Results