Revision as of 15:12, 28 October 2011

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 ([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.

Biobrick Number	Construct
[http://partsregistry.org/Part:BBa_K525121 K525121]	CspB with TAT-sequence and lipid anchor
[http://partsregistry.org/Part:BBa_K525123 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
[http://partsregistry.org/Part:BBa_K525121 K525121]	CspB with TAT-sequence and lipid anchor			X	X
[http://partsregistry.org/Part:BBa_K525123 K525123]	CspB without TAT-sequence and with lipid anchor			X	X

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 ([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.

Biobrick Number	Construct
[http://partsregistry.org/Part:BBa_K525222 K525222]	CspB without TAT-sequence and lipid anchor
[http://partsregistry.org/Part:BBa_K525223 K525223]	CspB without TAT-sequence and with lipid anchor
[http://partsregistry.org/Part:BBa_K525224 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
[http://partsregistry.org/Part:BBa_K525222 K525222]	CspB without TAT-sequence and lipid anchor	X	x		x
[http://partsregistry.org/Part:BBa_K525223 K525223]	CspB without TAT-sequence and with lipid anchor	x	x
[http://partsregistry.org/Part:BBa_K525224 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).

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

Characterization of expression and induction of [http://partsregistry.org/wiki/index.php/Part:BBa_K525305 K525305], a translational fusion of the sgsE gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K525303 K525303]) with mCitrine ([http://partsregistry.org/Part:BBa_J18931 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 [http://partsregistry.org/wiki/index.php/Part:BBa_K525405 K525405], a fusion of the sbpA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K525403 K525403]) with mCitrine ([http://partsregistry.org/Part:BBa_J18931 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.