Team:Bielefeld-Germany/Results/S-Layer

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(→The S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a)
 
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<html><img src="https://static.igem.org/mediawiki/2011/c/c7/Bielefeld-header-results-s-layer.png"/><p></p></html>
<html><img src="https://static.igem.org/mediawiki/2011/c/c7/Bielefeld-header-results-s-layer.png"/><p></p></html>
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=SgsE from ''Geobacillus stearothermophilus'' NRS 2004/3a=
 
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==Purification of SgsE fusion protein==
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=Summary of results=
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As seen in the analysis of the cultivations with expression of SgsE | mCitrine fusion proteins, these proteins form inclusion bodies in ''E. coli''. Inclusion bodies have the advantage that they are relatively easy to clean-up and are resistant to proteases. So the first purification step is to solve and set-free the inclusion bodies. This step is followed by two filtrations (300 kDa UF and 100 kDa DF/UF) to further concentrate and purify the S-layer proteins. After the filtrations, the remaining protein solution is dialized against ddH<sub>2</sub> for 18 h at 4 °C in the dark. The dialysis leads to a precipitation of the water-insoluble proteins. After centrifugation of the dialysate the water-soluble S-layer monomers remain in the supernatant and can be used for recrystallization experiments.  
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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<sup>-14</sup>). 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 fluorescence of the collected fractions of this purification strategy is shown in the following figure A:
 
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[[Image:Bielefeld-Germany2011-305_-_purificationfractions.jpg|700px|center|thumb|'''Fig. A: Fluorescence of collected fractions during purification of <partinfo>K525305</partinfo> fusion protein. ''']]
 
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A lot of protein is lost during the purification especially after centrifugation steps. The fluorescence in the urea containing fractions is lowered due to denaturation of the fluorescent protein. Some fluorescence could be regenerated by the recrystallization in HBSS. This purification strategy is very simple and can be carried out by nearly everyone in any lab being one first step to enable real do it yourself nanobiotechnology.  
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<center>
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<a href="https://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/CspB_CG"><img src="https://static.igem.org/mediawiki/2011/e/e4/Bielefeld2011_btn_CspB_CG.png" width="220px" style="padding:0px 0px 10px 0px;" /></a>
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<a href="https://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/CspB_CH"><img src="https://static.igem.org/mediawiki/2011/7/7f/Bielefeld2011_btn_CspB_CH.png" width="220px" style="padding:0px 0px 10px 0px;" /></a>
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<a href="https://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/SgsE"><img src="https://static.igem.org/mediawiki/2011/8/82/Bielefeld2011_btn_SgsE.png" width="220px" style="padding:0px 0px 10px 0px;" /></a>
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<a href="https://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/SbpA"><img src="https://static.igem.org/mediawiki/2011/8/83/Bielefeld2011_btn_SbpA.png" width="220px" style="padding:0px 0px 10px 0px;" /></a>
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</center>
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===Final purification strategy===
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'''<html><div align="center"><span style="font-size:18px;">Have you ever wanted to try nanobiotechnology yourself? <br>Just follow our GUIDE TO DIY NANOBIOTECHNOLOGY and take a look how it is done:</span style></div></html>'''
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Scheme of purification strategy for SgsE (fusion) proteins:
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[[Image:Bielefeld-Germany2011-305_405-Aufreinigung_symbol.png|800px|center]]
 
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First, SgsE is expressed in ''E. coli'' under the control of a T7 / lac promoter for separation of growth and production phase due to metabolic stress of the S-layer expression. Because the SgsE protein is forming inclusion bodies in ''E. coli'', an inclusion body purification with urea follows the cell lysis. The S-layers are further concentrated and purified by two ultrafiltration / diafiltration steps (300 kDa and 100 kDa) and afterwards dialysed against water leading to the precipitation of water-insoluble proteins. The supernatant contains the monomeric SgsE solution.
 
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[[Team:Bielefeld-Germany/Protocols/Downstream-processing#Fusion_proteins_of_SgsE_and_SbpA | Click for detailed information]]
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<a href="https://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/Guide"><img src="https://static.igem.org/mediawiki/2011/5/56/Bielefeld2011_Guide.png" width="350px" style="padding:0px 0px 10px 0px;" /></a>
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</html>
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</center>
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==Immobilization behaviour==
 
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After purification, solutions of monomeric SgsE S-layer proteins can be recrystallized and immobilized on silicon dioxide beads in HBSS (Hank's buffered saline solution). After the recrystallization procedure the beads are washed with and stored in ddH<sub>2</sub>O at 4 °C in the dark. The fluorescence of the collected fractions of a recrystallization experiment with <partinfo>K525305</partinfo> are shown in fig. X. 100 mg beads were coated with 100 µg of protein. The figure shows, that not all of the protein is immobilized on the beads (supernatant fraction) but the immobilization is pretty stable (very low fluorescence in the wash). After the immobilization, the beads show a high fluorescence indicating the binding of the SgsE | mCitrine fusion protein.
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==The S-layer protein PS2 of ''Corynebacterium glutamicum''==
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[[Image:Bielefeld-Germany2011-305_100-fractions.jpg|600px|center|thumb|'''Fig. X: Measured fluorescence of collected fractions of immobilization of purified <partinfo>K525305</partinfo> on silica dioxide beads (n = 3, 100 mg mL<sup>-1</sup> SiO<sub>2</sub>, time of recrystallization: 4 h). ''']]
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* 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.
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<center>
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{|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"
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|-
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!style="border-style: solid; border-width: 0 1px 1px 0"| BioBrick Number 
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!style="border-style: solid; border-width: 0 0 1px 0"| Construct
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|-
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|style="border-style: solid; border-width: 0 1px 0 0"| [http://partsregistry.org/Part:BBa_K525121 K525121]
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|style="border-style: solid; border-width: 0"| [[Team:Bielefeld-Germany/Results/S-Layer/CspB_CG#CspB_with_TAT-sequence_and_lipid_anchor|<html>&nbsp;</html>CspB with TAT-sequence and lipid anchor<html>&nbsp;</html>]]
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|
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|-
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|style="border-style: solid; border-width: 0 1px 0 0"| [http://partsregistry.org/Part:BBa_K525123 K525123]
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|style="border-style: solid; border-width: 0"| [[Team:Bielefeld-Germany/Results/S-Layer/CspB_CG#CspB_without_TAT-sequence_and_with_lipid_anchor|<html>&nbsp;</html>CspB without TAT-sequence and with lipid anchor<html>&nbsp;</html>]]
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|}
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</center>
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===Optimal bead to protein ratio for immobilization===
 
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To determine the optimal ratio of silica beads to protein for immobilization, the degree of clearance ϕ<sub>C</sub> in the supernatant is calculated and plotted against the concentration of silica beads used in the accordant immobilization experiment (compare fig. A):
 
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* 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 ddH<sub>2</sub>O, as well as the fractions after treating the pellet from lysis with different detergents.
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[[Image:Bielefeld-Germany2011-degreeofclearanceformula.png|150px|center]] <div align="right">(2)</div>
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* 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.
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<center>
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The data was collected in three indipendent experiments. The fluorescence of the samples was measured in the supernatant of the immobilization experiment after centrifuging the silica beads. The fluorescence of the control was measured in a sample which was treated exactly like the others but no silica beads were added. 100 µg protein was used for one immobilization experiment. The data was fitted with a sigmoidal dose-response function of the form
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{|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"
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|-
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!style="border-style: solid; border-width: 0 1px 1px 0"| <html>&nbsp;</html>Biobrick Number<html>&nbsp;</html>
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!style="border-style: solid; border-width: 0 1px 1px 0"| Construct
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!style="border-style: solid; border-width: 0 1px 1px 0"| <html>&nbsp;</html>Supernatant<html>&nbsp;</html>
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!style="border-style: solid; border-width: 0 1px 1px 0"| <html>&nbsp;</html>Periplasm<html>&nbsp;</html>
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!style="border-style: solid; border-width: 0 1px 1px 0"| <html>&nbsp;</html>Cell lysis<html>&nbsp;</html>
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!style="border-style: solid; border-width: 0 1px 1px 0"| <html>&nbsp;</html>Integration in cell membrane<html>&nbsp;&nbsp;</html>
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!style="border-style: solid; border-width: 0 0 1px 0"| Inclusion bodies<html>&nbsp;</html>
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|-
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|style="border-style: solid; border-width: 0 1px 0 0"| [http://partsregistry.org/Part:BBa_K525121 K525121]
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|style="border-style: solid; border-width: 0 1px 0 0"| [[Team:Bielefeld-Germany/Results/S-Layer/CspB_CG#Identification_and_localisation|<html>&nbsp;</html>CspB with TAT-sequence and lipid anchor]]
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|style="border-style: solid; border-width: 0 1px 0 0"|
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|style="border-style: solid; border-width: 0 1px 0 0"|
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|style="border-style: solid; border-width: 0 1px 0 0"| '''<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>X'''
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|style="border-style: solid; border-width: 0 1px 0 0"| '''<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>X'''
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|style="border-style: solid; border-width: 0 0 0 0"|
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|
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|-
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|style="border-style: solid; border-width: 0 1px 0 0"| [http://partsregistry.org/Part:BBa_K525123 K525123]
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|style="border-style: solid; border-width: 0 1px 0 0"| [[Team:Bielefeld-Germany/Results/S-Layer/CspB_CG#CspB_without_TAT-sequence_and_with_lipid_anchor|<html>&nbsp;</html>CspB without TAT-sequence and with lipid anchor <html>&nbsp;</html>]]
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|style="border-style: solid; border-width: 0 1px 0 0"|
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|style="border-style: solid; border-width: 0 1px 0 0"|
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|style="border-style: solid; border-width: 0 1px 0 0"| '''<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>X'''
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|style="border-style: solid; border-width: 0 1px 0 0"| '''<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>X'''
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|style="border-style: solid; border-width: 0 0 0 0"|
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|}
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</center>
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[[Image:Bielefeld_Doseresponse_fit.jpg|175px|center]] <div align="right">(3)</div>
 
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<a href="https://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/CspB_CG"><img src="https://static.igem.org/mediawiki/2011/e/e4/Bielefeld2011_btn_CspB_CG.png" width="220px" style="padding:0px 0px 10px 0px;" /></a>
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</html>
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</center>
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with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x<sub>0</sub>) (R² = 0.874).
 
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The fit indicates that a good silica concentration for 100 µg of protein is 150 - 200 mg mL<sup>-1</sup>. This set-up leads to saturated beads with low waste of protein. So a good protein / bead ratio to work with is 5 - 7 * 10<sup>-4</sup>.
 
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[[Image:Bielefeld-Germany2011-degreeofclearance305.jpg|700px|center|thumb|'''Fig. A: Degree of clearance of the fluorescence in the supernatant plotted against the concentration of silicium dioxide beads used to immobilize <partinfo>K525305</partinfo> (n = 3). Data is fitted with dose-reponse function (R² = 0.874). ''']]
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<!-- <html><div style="font-size:30px; text-align:center; font-weight:bold;"><a href="https://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/CspB_CG">Complete Results</a></div></html> -->
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==The S-layer protein PS2 of ''Corynebacterium 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 view the results.
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<center>
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=SbpA from ''Lysinbacillus sphaericus'' CCM 2177=
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{|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"
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|-
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!style="border-style: solid; border-width: 0 1px 1px 0"| BioBrick Number 
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!style="border-style: solid; border-width: 0 0 1px 0"| Construct
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|-
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|style="border-style: solid; border-width: 0 1px 0 0"| [http://partsregistry.org/Part:BBa_K525222 K525222]
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|style="border-style: solid; border-width: 0"| [[Team:Bielefeld-Germany/Results/S-Layer/CspB_CH#CspB_without_TAT-sequence_and_lipid_anchor|<html>&nbsp;</html>CspB without TAT-sequence and lipid anchor]]
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|
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|-
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|style="border-style: solid; border-width: 0 1px 0 0"| [http://partsregistry.org/Part:BBa_K525223 K525223]
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|style="border-style: solid; border-width: 0"| [[Team:Bielefeld-Germany/Results/S-Layer/CspB_CH#CspB_without_TAT-sequence_and_with_lipid_anchor|<html>&nbsp;</html>CspB without TAT-sequence and with lipid anchor]]
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|
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|-
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|style="border-style: solid; border-width: 0 1px 0 0"| [http://partsregistry.org/Part:BBa_K525224 K525224]
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|style="border-style: solid; border-width: 0"| [[Team:Bielefeld-Germany/Results/S-Layer/CspB_CH#CspB_with_TAT-sequence_and_without_lipid_anchor|<html>&nbsp;</html>CspB with TAT-sequence and without lipid anchor]]
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|}
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</center>
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==Purification of SbpA fusion protein==
 
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As seen in the analysis of the cultivations with expression of SbpA | mCitrine fusion proteins, these proteins form inclusion bodies in ''E. coli''. Inclusion bodies have the advantage that they are relatively easy to clean-up and are resistant to proteases. So the first purification step is to solve and set-free the inclusion bodies. This step is followed by two filtrations (300 kDa UF and 100 kDa DF/UF) to further concentrate and purify the S-layer proteins. After the filtrations, the remaining protein solution is dialized against ddH<sub>2</sub> for 18 h at 4 °C in the dark. The dialysis leads to a precipitation of the water-insoluble proteins. After centrifugation of the dialysate the water-soluble S-layer monomers remain in the supernatant and can be used for recrystallization experiments.
 
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The fluorescence of some collected, important fractions of this purification strategy is shown in the following figure A:
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* 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 ddH<sub>2</sub>O, as well as the fractions after treating the pellet from lysis with different detergents.
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[[Image:Bielefeld-Germany2011-405-purificationfractions.jpg|700px|center|thumb|Fig. A: '''Fluorescence of collected fractions during purification of <partinfo>K525405</partinfo> fusion protein. Abbreviations: pell.: pellet, s.n.: supernatant, ret.: retentate, perm.: permeate.''']]
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* 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.
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A lot of protein is lost during the purification especially after centrifugation steps (compared to filtrations). The fluorescence in the urea containing fractions is lowered due to denaturation of the fluorescent protein. This purification strategy is very simple and can be carried out by nearly everyone in any lab being one first step to enable real do it yourself nanobiotechnology.
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<center>
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{|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding: 0"
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|-
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!style="border-style: solid; border-width: 0 1px 1px 0"|  <html>&nbsp;</html>BioBrick Number<html>&nbsp;</html>
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!style="border-style: solid; border-width: 0 1px 1px 0"| Construct
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!style="border-style: solid; border-width: 0 1px 1px 0"| <html>&nbsp;</html>Supernatant<html>&nbsp;</html>
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!style="border-style: solid; border-width: 0 1px 1px 0"| <html>&nbsp;</html>Periplasm<html>&nbsp;</html>
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!style="border-style: solid; border-width: 0 1px 1px 0"| <html>&nbsp;</html>Lysate<html>&nbsp;</html>
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!style="border-style: solid; border-width: 0 1px 1px 0"| <html>&nbsp;</html>Integration in cell membrane<html>&nbsp;</html>
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!style="border-style: solid; border-width: 0 0 1px 0"| <html>&nbsp;</html>Inclusion bodies
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|-
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|style="border-style: solid; border-width: 0 1px 0 0"| [http://partsregistry.org/Part:BBa_K525222 K525222]
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|style="border-style: solid; border-width: 0 1px 0 0"| [[Team:Bielefeld-Germany/Results/S-Layer/CspB_CH#Identification_and_localisation|<html>&nbsp;</html>CspB without TAT-sequence and lipid anchor<html>&nbsp;</html>]]
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|style="border-style: solid; border-width: 0 1px 0 0"|  <html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>'''X'''
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|style="border-style: solid; border-width: 0 1px 0 0"| <html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>x
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|style="border-style: solid; border-width: 0 1px 0 0"|
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|style="border-style: solid; border-width: 0 1px 0 0"| <html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>x
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|style="border-style: solid; border-width: 0 0 0 0"|
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|
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|-
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|style="border-style: solid; border-width: 0 1px 0 0"| [http://partsregistry.org/Part:BBa_K525223 K525223]
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|style="border-style: solid; border-width: 0 1px 0 0"| [[Team:Bielefeld-Germany/Results/S-Layer/CspB_CH#Identification_and_localisation_2|<html>&nbsp;</html>CspB without TAT-sequence and with lipid anchor<html>&nbsp;</html>]]
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|style="border-style: solid; border-width: 0 1px 0 0"| <html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>x
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|style="border-style: solid; border-width: 0 1px 0 0"| <html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>x
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|style="border-style: solid; border-width: 0 1px 0 0"|
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|style="border-style: solid; border-width: 0 1px 0 0"|
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|style="border-style: solid; border-width: 0 0 0 0"|
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|
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|-
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|style="border-style: solid; border-width: 0 1px 0 0"| [http://partsregistry.org/Part:BBa_K525224 K525224]
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|style="border-style: solid; border-width: 0 1px 0 0"| [[Team:Bielefeld-Germany/Results/S-Layer/CspB_CH#Identification_and_localisation_3|<html>&nbsp;</html>CspB with TAT-sequence and without lipid anchor<html>&nbsp;</html>]]
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|style="border-style: solid; border-width: 0 1px 0 0"| <html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>'''X'''
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|style="border-style: solid; border-width: 0 1px 0 0"| <html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>x
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|style="border-style: solid; border-width: 0 1px 0 0"| <html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>x
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|style="border-style: solid; border-width: 0 1px 0 0"|
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|style="border-style: solid; border-width: 0 0 0 0"|
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|}
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</center>
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===Final purification strategy===
 
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Scheme of purification strategy for SbpA (fusion) proteins:
 
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[[Image:Bielefeld-Germany2011-305_405-Aufreinigung_symbol.png|800px|center]]
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* 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: [[Team:Bielefeld-Germany/Results/S-Layer/CspB_CH#Purification|Purification and final strategy]]).
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First, SbpA is expressed in ''E. coli'' under the control of a T7 / lac promoter for separation of growth and production phase due to metabolic stress of the S-layer expression. Because the SbpA protein is forming inclusion bodies in ''E. coli'', an inclusion body purification with urea follows the cell lysis. The S-layers are further concentrated and purified by two ultrafiltration / diafiltration steps (300 kDa and 100 kDa) and afterwards dialysed against water leading to the precipitation of water-insoluble proteins. The supernatant contains the monomeric SbpA solution.
 
-
[[Team:Bielefeld-Germany/Protocols/Downstream-processing#Fusion_proteins_of_SgsE_and_SbpA | Click for detailed information]]
+
<!-- <html><div style="font-size:30px; text-align:center; font-weight:bold;"><a href="https://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/CspB_CH">Complete Results</a></div></html> -->
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<center>
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<html>
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<a href="https://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/CspB_CH"><img src="https://static.igem.org/mediawiki/2011/7/7f/Bielefeld2011_btn_CspB_CH.png" width="220px" style="padding:0px 0px 10px 0px;" /></a>
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</html>
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==Immobilization behaviour==
 
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After purification, solutions of monomeric SbpA S-layer proteins can be recrystallized and immobilized on silicon dioxide beads in recrystallization buffer (0.5 mM Tris-HCl, pH 9, 10 mM CaCl<sub>2</sub>). After the recrystallization procedure the beads are washed with and stored in ddH<sub>2</sub>O at 4 °C in the dark. The fluorescence of the collected fractions of a recrystallization experiment with <partinfo>K525405</partinfo> are shown in fig. X. 100 mg beads were coated with 100 µg of protein. The figure shows, that not all of the protein is immobilized on the beads (supernatant fraction) but the immobilization is pretty stable (very low fluorescence in the wash). After the immobilization, the beads show a high fluorescence indicating the binding of the SbpA | mCitrine fusion protein.
 
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[[Image:Bielefeld-Germany2011-405_100-fractions.jpg|600px|center|thumb|'''Fig. X: Measured fluorescence of collected fractions of immobilization of purified <partinfo>K525405</partinfo> on silica dioxide beads (n = 3, 100 mg mL<sup>-1</sup> SiO<sub>2</sub>, time of recrystallization: 4 h). ''']]
 
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</center>
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==The S-layer protein SgsE of ''Geobacillus stearothermophilus'' NRS 2004/3a==
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===Optimal bead to protein ratio for immobilization===
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* 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: [[Team:Bielefeld-Germany/Results/S-Layer/SgsE#Expression_in_E._coli|Expression in ''E. coli'']]).
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To determine the optimal ratio of silica beads to protein for immobilization, the degree of clearance ϕ<sub>C</sub> in the supernatant is calculated and plotted against the concentration of silica beads used in the accordant immobilization experiment (compare fig. A):  
+
 +
* 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: [[Team:Bielefeld-Germany/Results/S-Layer/SgsE#Purification_of_SgsE_fusion_protein|Purification of SgsE fusion protein inclusion bodies]])
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[[Image:Bielefeld-Germany2011-degreeofclearanceformula.png|150px|center]] <div align="right">(2)</div>
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* Fusing SgsE gen with His-tag and simple purification by using a [https://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/SgsE#Purification_of_SgsE_.7C_mCitrine_with_His-tag 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: [https://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/SgsE#Purification_of_SgsE_.7C_mCitrine_with_His-tag 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.
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The data was collected in three indipendent experiments. The fluorescence of the samples was measured in the supernatant of the immobilization experiment after centrifuging the silica beads. The fluorescence of the control was measured in a sample which was treated exactly like the others but no silica beads were added. 100 µg protein was used for one immobilization experiment. The data was fitted with a sigmoidal dose-response function of the form
+
* Characterization of immobilization behaviour of proteins used to coat silica beads through measurement of fluorescence of supernatant, wash fraction and beads (See: [[Team:Bielefeld-Germany/Results/S-Layer/SgsE#Immobilization_behaviour|Immobilization behaviour]]).
 +
* Immobilization experiments to determine the optimal bead to protein ratio (See: [[Team:Bielefeld-Germany/Results/S-Layer/SgsE#Optimal_bead_to_protein_ratio_for_immobilization|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&nbsp;mL<sup>-1</sup>.
-
[[Image:Bielefeld_Doseresponse_fit.jpg|175px|center]] <div align="right">(3)</div>
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* Expression and purification of an SgsE | luciferase fusion protein
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<!-- <html><div style="font-size:30px; text-align:center; font-weight:bold;"><a href="https://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/SgsE">Complete Results</a></div></html> -->
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with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x<sub>0</sub>) (R² = 0.997).
 
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The fit indicates that a good silica concentration for 100 µg of protein is 200 - 250 mg mL<sup>-1</sup>. This set-up leads to saturated beads with low waste of protein. So a good protein / bead ratio to work with is 7 - 9 * 10<sup>-4</sup>.
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<center>
 +
<html>
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<a href="https://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/SgsE"><img src="https://static.igem.org/mediawiki/2011/8/82/Bielefeld2011_btn_SgsE.png" width="220px" style="padding:0px 0px 10px 0px;" /></a>
 +
</html>
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[[Image:Bielefeld-Germany2011-degreeofclearance405.jpg|700px|center|thumb|'''Fig. A: Degree of clearance of the fluorescence in the supernatant plotted against the concentration of silicium dioxide beads used to immobilize <partinfo>K525405</partinfo> (n = 3). Data is fitted with dose-reponse function (R² = 0.997). ''']]
 
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=CspB from ''Brevibacterium flavum''=
 
 +
</center>
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==The S-layer protein SbpA of ''Lysinibacillus sphaericus'' CCM 2177==
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=CspB from ''Corynebacterium halotolerans''=
+
* 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: [[Team:Bielefeld-Germany/Results/S-Layer/SbpA#Expression_in_E._coli|Expression in ''E. coli'']]).
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==CspB with TAT-sequence and without lipid anchor==
+
* 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: [[Team:Bielefeld-Germany/Results/S-Layer/SbpA#Purification_of_SbpA_fusion_protein|Purification of SbpA fusion protein inclusion bodies]]).
-
===Purification===
+
* Immobilization experiments to determine optimal bead to protein ratio (See: [[Team:Bielefeld-Germany/Results/S-Layer/SbpA#Immobilization_behaviour|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&nbsp;mL<sup>-1</sup>.
 +
<!-- <html><div style="font-size:30px; text-align:center; font-weight:bold;"><a href="https://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/SbpA">Complete Results</a></div></html> -->
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After the localisation of the S-layer protein in ''E. coli'', different methods for purification were tested. The results of these methods are shown in fig. X. Fig. X shows, that the CspB protein does not form inclusion bodies in ''E. coli'' and most of the protein is transported out of the cell into the periplasm and a lot of protein is even secreted into the medium (all fractions were concentrated by filtration and precipitation, respectively). The secretion into the culture medium is very interesting because the purification is much faster (no cell disruption necessary).
 
-
[[Image:Bielefeld-Germany2011-CH4-purificationfractions.jpg|700px|center|thumb|'''Fig. X: Fluorescence of collected fractions of different methods to release and concentrate <partinfo>K525234</partinfo> protein from a cultivation in ''E. coli''. ''']]
 
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The highest fluorescence could be obtained by a precipitation with ammonium sulfate of the culture supernatant followed by an ultrafiltration with a 300 kDa membrane and a diafiltration with a 50 kDa membrane. The diafiltration was against a binding buffer for an anion exchange chromatography (25 mM sodium acetate, 25 mM sodium chloride) with pH 6, due to the theoretical pI of <partinfo>k525234</partinfo>. The fluorescence of the collected fractions of the following anion exchange chromatography are shown in fig. B.  
+
<center>
 +
<html>
 +
<a href="https://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/SbpA"><img src="https://static.igem.org/mediawiki/2011/8/83/Bielefeld2011_btn_SbpA.png" width="220px" style="padding:0px 0px 10px 0px;" /></a>
 +
</html>
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[[Image:Bielefeld-Germany2011-CH4_Med_IEX.jpg|700px|center|thumb|'''Fig. B: Fluorescence of collected fractions of an anion exchange chromatography of <partinfo>K525234</partinfo> after concentration from the culture supernatant. ''']]
 
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The binding conditions are well chosen because nearly all of the protein binds to the column. The protein is eluted from the column with rising sodium chloride concentrations. The highest fluorescence is in the elution fraction with 400 mM sodium chloride.  600 mM sodium chloride elutes all of the S-layer fusion proteins.
 
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====Final purification strategy===
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</center>

Latest revision as of 03:44, 29 October 2011


Contents

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   Inclusion bodies
[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)
  • 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.




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