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Team:Bielefeld-CeBiTec/Results/CO2-fixation/Carboxysome
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<h6>Carboxysome purification</h6> | <h6>Carboxysome purification</h6> | ||
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| - | Purification of recombinant proteins is one essential foundation of syntetic biology. For this reason, it was our approach to purify the carboxysome from the chemolitoautotroph model organism <i>Halothiobacillus neapolitanus</i>, recombinant expressed in <i>E. coli</i>. Our work for purifying the carboxysome was based on the plasmide | + | Purification of recombinant proteins is one essential foundation of syntetic biology. For this reason, it was our approach to purify the carboxysome from the chemolitoautotroph model organism <i>Halothiobacillus neapolitanus</i>, recombinant expressed in <i>E. coli</i>. Our work for purifying the carboxysome was based on the plasmide pHnBCcsoS1D, which we received from addgene. |
| - | For cultivation and recombinant expression of the carboxysome, we followed the method for cultivation described in <a href="#bonacchi2012">Bonacchi et al., 2012</a> and the method for purification described by <a href="#so2004">So et al., 2004</a>. To proof the expression of the carboxysomal proteins, we analysed recombinant protein expression by <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis%20%28SDS-PAGE%29" target="_blank">SDS-PAGE </a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Matrix-assistedLaserDesorption/Ionization%E2%80%93Timeofflight%20%28MALDI-TOF%29" target="_blank">MALDI-TOF</a>. The results of the SDS-PAGE are shown in Figure : | + | For cultivation and recombinant expression of the carboxysome, we followed the method for cultivation described in <a href="#bonacchi2012">Bonacchi et al., 2012</a> and the method for purification described by <a href="#so2004">So et al., 2004</a>. To proof the expression of the carboxysomal proteins, we analysed recombinant protein expression by <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis%20%28SDS-PAGE%29" target="_blank">SDS-PAGE </a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Matrix-assistedLaserDesorption/Ionization%E2%80%93Timeofflight%20%28MALDI-TOF%29" target="_blank">MALDI-TOF</a>. The results of the SDS-PAGE are shown in Figure 1-3: |
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<a href="https://static.igem.org/mediawiki/2014/e/ec/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_0.5_mM.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/e/ec/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_0.5_mM.jpg" width="220px"></a><br> | <a href="https://static.igem.org/mediawiki/2014/e/ec/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_0.5_mM.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/e/ec/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_0.5_mM.jpg" width="220px"></a><br> | ||
| - | <font size="1" style="text-align:center;">Proteinexpression of pHnCBcsoS1D <br>induced with 0.5 mM IPTG</font> | + | <font size="1" style="text-align:center;"><b>Figure 1:<b>Proteinexpression of pHnCBcsoS1D <br>induced with 0.5 mM IPTG</font> |
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<a href="https://static.igem.org/mediawiki/2014/c/cf/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_2_mM.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/c/cf/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_2_mM.jpg" width="220px"></a><br> | <a href="https://static.igem.org/mediawiki/2014/c/cf/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_2_mM.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/c/cf/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_2_mM.jpg" width="220px"></a><br> | ||
| - | <font size="1" style="text-align:center;">Proteinexpression of pHnCBcsoS1D <br>induced with 2 mM IPTG</font> | + | <font size="1" style="text-align:center;"><b>Figure 2:<b> Proteinexpression of pHnCBcsoS1D <br>induced with 2 mM IPTG</font> |
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<a href="https://static.igem.org/mediawiki/2014/a/af/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_5_mM.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/a/af/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_5_mM.jpg" width="220px"></a><br> | <a href="https://static.igem.org/mediawiki/2014/a/af/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_5_mM.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/a/af/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_5_mM.jpg" width="220px"></a><br> | ||
| - | <font size="1" style="text-align:center;">Proteinexpression of pHnCBcsoS1D <br>induced with 5 mM IPTG</font> | + | <font size="1" style="text-align:center;"><b>Figure 3:<b> Proteinexpression of pHnCBcsoS1D <br>induced with 5 mM IPTG</font> |
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Revision as of 19:44, 16 October 2014
CO2 fixation
Abstract
The expression of BBa_K1465223 (pSB1C3-T7:sap-csoS4AB-csoS1CA:gfp-csoS1B) in E. coli leads to the assembly of carboxysomes. We used a translational fusion of one shell protein with the CDS of the green fluorescent protein as an indicator of correct protein folding. The observed fluorescence in our E.coli cells is concentrated at different points and indicates the presence of functional carboxysomes.
Introduction
In nature there are plasmids which encode different proteins of the carboxysome in bacteria. One such plasmid is pHnCBS1D which was found in Halothiobacillus neapolitanis (Cannon and Shively, 1983). Different parts of this plasmid were used for the production of BioBricks. These BioBricks were used for the construction of a synthetic carboxysome-encoding plasmid. This bottom up approach allows to verify the essentiallity of the different components. We used a translational fusion of csoS1A and gfp (BBa_K1465222) as indicator of correct protein folding. A concentrated subcellular localisation ot the fluorescence shows the positions of carboxysoms. This reporter function of GFP was identified and used before (Waldo et al., 1999; Hsu et al., 2009).
Shell protein folding
The expression of csoS1A:gfp with all other known essentiell shell proteins (BBa_K1465222) did not yield any fluorescence (VERWEIS auf Ergebnisse von Birte). This indicates that the presence of a shell assosiated protein (SAP) encoded by csoS2 is needed for the correct folding of the shell proteins and the assembly of the whole carboysome. The combined expression of the sap and the cso coding sequences (BBa_K1465223) yields green fluorescence. This fluorescence was quantified by photometric measurements (Link auf Methode und auf Ergebnisse). The signal intensity of the E. coli KRX wildtype (negative control) was compared to cells carrying either a plasmid with (BBa_K1465223) or without sap(BBa_K1465222). E. coli KRX cells with pSB1C3-pTET:gfp (BBa_I13522) served as a positive control. From these results we deduce the correct folding of the shell proteins in presence of the shell assosiated protein.
[Tabelle / Grafik mit Ergebnissen vom GloMax]
Carboxysome assembly
To show the successfull assembly of the carboxysom we analysed the subcellular localisation of the green fluorescent protein. Pictures taken through a fluorescence microscope are shown in figure XXXX (fig = Mikroskopfotos von Birte). We assume that the concentrated green fluorescence results from GFP molecules which are fused to shell proteins of a successfully assembled carboxysome.
[pictures]
This assembly was possible without the expression of csoS1D another coding sequence which is located on pHnCBS1D. The resulting protein is probably responsible for the pore size in the carboxysome envelope (Kinney et al., 2011; Bonacchi et al., 2012). Nevertheless it seems that it has no essentiell function in protein folding.
[mikroskopische Aufnahmen => ggf. Evolution der Mikroskope]
Carboxysome application
The functional carboxysom could be used for the carbon dioxide fixation. Therefore three enzymes needs to be heterologous expressed in E. coli. We tried to combine the carboxysome encoding genes and two of the required enzymes on a single plasmid. One RubisCO (BBa_K1465202) and one phosphoribulokinase (BBa_K1465201) encoding sequence should be integrated upstream of the carboxysomal operon.
Summary and outlook
We report the construction of a synthetic plasmid encoding a functional carboxysome. Despite the fact that he have not shown the fixation of carbon dioxide in this compartiment yet we would like to emphasise the construction of a usefull microcompartment. This could be used for anarobic reactions in aerobic cultures of E.coli. One possible application beside the carbon dioxide fixation could be the fixation of atmospheric nitrogen which is essentiell for the growth of plants.
Carboxysome purification
Purification of recombinant proteins is one essential foundation of syntetic biology. For this reason, it was our approach to purify the carboxysome from the chemolitoautotroph model organism Halothiobacillus neapolitanus, recombinant expressed in E. coli. Our work for purifying the carboxysome was based on the plasmide pHnBCcsoS1D, which we received from addgene. For cultivation and recombinant expression of the carboxysome, we followed the method for cultivation described in Bonacchi et al., 2012 and the method for purification described by So et al., 2004. To proof the expression of the carboxysomal proteins, we analysed recombinant protein expression by SDS-PAGE and MALDI-TOF. The results of the SDS-PAGE are shown in Figure 1-3:
 Figure 1:Proteinexpression of pHnCBcsoS1D induced with 0.5 mM IPTG |
 Figure 2: Proteinexpression of pHnCBcsoS1D induced with 2 mM IPTG |
 Figure 3: Proteinexpression of pHnCBcsoS1D induced with 5 mM IPTG |
References
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Cannon, Shively 1983. Characterisation of a Homogenous Preparation of Carboxysomes from Thiobacillus neapolitanus. Archive of Microbiology, vol. 134, pp. 52-59
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Waldo, G. S., B. M. Standish, J. Berendzen, und T. C. Terwilliger. „Rapid Protein-Folding Assay Using Green Fluorescent Protein“. Nature Biotechnology 17, Nr. 7 (Juli 1999): 691–95. doi:10.1038/10904.
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Hsu, Shang-Te Danny, Georg Blaser, und Sophie E. Jackson. „The Folding, Stability and Conformational Dynamics of Beta-Barrel Fluorescent Proteins“. Chemical Society Reviews 38, Nr. 10 (2009): 2951–65. doi:10.1039/b908170b.
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Bonacci, Walter, Poh K. Teng, Bruno Afonso, Henrike Niederholtmeyer, Patricia Grob, Pamela A. Silver, und David F. Savage. „Modularity of a Carbon-Fixing Protein Organelle“. Proceedings of the National Academy of Sciences 109, Nr. 2 (1. Oktober 2012): 478–83. doi:10.1073/pnas.1108557109.
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Kinney, James N., Seth D. Axen, und Cheryl A. Kerfeld. „Comparative analysis of carboxysome shell proteins“. Photosynthesis Research 109, Nr. 1–3 (September 2011): 21–32. doi:10.1007/s11120-011-9624-6.
