From 2011.igem.org

Summary of results

Four different S-layer BioBricks with various lattice structures were created and sent to the Partsregistry. The behaviour of these genes when expressed in E. coli was characterized and easy to perform 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 expression with a TAT-sequence and a lipid anchor resulted in an integration into the cell membrane, whereas the expression with a TAT-sequence alone resulted in a secretion into the medium. We also detected that those S-layers seem to stabilize the biological active conformation of mRFP. Furthermore, we expressed and purified a fluorescent CspB fusion protein from C. halotolerans which has not been expressed in E. coli before.

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The S-layer protein PS2 of Corynebacterium 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 view 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 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 Corynebacterium 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 view 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.

Localization of fusion protein in the cell by fractionation with different detergents and periplasmatic disruption. Identification of protein-containing fractions by fluorescence measurements and MALDI-TOF analysis. Table below shows the supposed location of the fusion protein in the cell, depending on presence of lipid anchor and TAT-sequence. Small x means a small amount of protein, large X means that the main amount of protein was found in this fraction. Click on the construct to show the results.

BioBrick Number	Construct	Supernatant	Periplasm	Lysate	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 gene ([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. Provision of methods to quickly obtain water-soluble fusion protein monomers for recrystallisation and coating through dialyzation (See: Purification of SgsE fusion protein inclusion bodies)

Fusing SgsE gen with His-tag and simple purification by using a denaturating His-tag affinity chromatography. This strategie has the advantage that no time-consuming and complex inclusion body purification and filtration is necessary. Additional to the simplification a higher purity could of the S-layer protein could be reached. (See: Purification of SgsE | mCitrine with His-tag)

Expression of SgsE|firefly luciferase [http://partsregistry.org/Part:BBa_K525311 (K525311)] fusion protein under the control of a T7 / lac promoter in E. coli KRX.

Characterization of immobilization behaviour of proteins used to coat silica beads through measurement of fluorescence of supernatant, wash fraction and beads (See: Immobilization behaviour).

Immobilization experiments 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 concentration for immobilization of 100 µg protein could be calculated to be approx. 150 - 200 mg mL^-1.

Expression and purification of an SgsE | luciferase fusion protein

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 translational fusion of the sbpA gene ([http://partsregistry.org/wiki/index.php/Part:BBa_K525403 K525403]) with the mCitrine gene ([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. Provision of methods to easily obtain water-soluble fusion protein monomers for recrystallization and coating experiments through dialysis (See: Purification of SbpA fusion protein inclusion bodies).

Immobilization experiments 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 approx. 200 - 250 mg mL^-1.

Team:Bielefeld-Germany/Results/S-Layer