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Team:Bielefeld-CeBiTec/Results/CO2-fixation/Carboxysome
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| - | In nature there are plasmids which encode different proteins of the carboxysome in bacteria. One such plasmid is <a href="2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#pHnCBS1D"><i>pHnCBS1D</i></a> which was found in <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#H.neapolitanus"><i>Halothiobacillus neapolitanis</i></a> (<a href="#cannon1983">Cannon and Shively, 1983</a>). Different parts of this plasmid were used for the production of BioBricks. <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#H.neapolitanus">These BioBricks</a> 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 <i>csoS1A</i> and <i>gfp</i> (<a href="http://parts.igem.org/Part:BBa_K1465222">BBa_K1465222</a>) 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 ( | + | In nature there are plasmids which encode different proteins of the carboxysome in bacteria. One such plasmid is <a href="2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#pHnCBS1D"><i>pHnCBS1D</i></a> which was found in <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#H.neapolitanus"><i>Halothiobacillus neapolitanis</i></a> (<a href="#cannon1983">Cannon and Shively, 1983</a>). Different parts of this plasmid were used for the production of BioBricks. <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#H.neapolitanus">These BioBricks</a> 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 <i>csoS1A</i> and <i>gfp</i> (<a href="http://parts.igem.org/Part:BBa_K1465222">BBa_K1465222</a>) 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 (<a href="#waldo1999">Waldo et al., 1999</a>). |
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| + | Waldo, G. S., B. M. Standish, J. Berendzen, und T. C. Terwilliger. „Rapid Protein-Folding Assay Using Green Fluorescent Protein“. <a href="http://www.nature.com/nbt/journal/v17/n7/full/nbt0799_691.html">Nature Biotechnology</a> 17, Nr. 7 (Juli 1999): 691–95. doi:10.1038/10904. | ||
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Revision as of 23:43, 15 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).
Shell protein folding
The expression of csoS1A:gfp with all other known essentiell shell proteins (BioBrickNummer von pSB1C3-cso:gfp + link) did not yield any fluorescence (VERWEIS auf Ergebnisse von Birte). This indicates that the presence of a shell assosiated protein (SAP) 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 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 comparted to cells carrying either a plasmid with or without sap. E. coli KRX cells with pSB1C3-pTET:gfp (BB-Nummer einsetzen+Link) 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. 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 (REFERENZ). Nevertheless it seems that it has no essentiell function. [mikroskopische Aufnahmen => ggf. Evolution der Mikroskope]
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.
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.
