Team:Bielefeld-CeBiTec/Results/CO2-fixation/Carboxysome

From 2014.igem.org

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The expression of pSB1C3-T7:sap-csoS4AB-csoS1CA:gfp-csoS1B in <i>E. coli</i> leads to the assembly of <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Project/CO2-fixation/Carboxysome">carboxysomes</a>. 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 <i>E.coli</i> cells is concentrated at different points and indicates the presence of functional carboxysomes.
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The carboxysome is a protein-enveloped microcompartement for the carbon fixation in cyanobacteria like <i>Synechococus elangatus</i> or sulfur bacteria like <i>Halothiobacillus neapolitanus</i>. It is composed of thousands of subunits including hexameric and pentameric proteins that form a shell to encapsulate the enzymes Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and the carbonic anhydrase. The compartimentalization allows an incrasing substrate concentration for the RubisCO within this microcompartement and therefore an improved catalytic efficiency of the encapsulated enzymes.<br>
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First of all carbonate (HCO<sub>3</sub><sup>-</sup>) diffuse into the caboxysome and is than converted into carbon dioxide (CO<sub>2</sub>) enabling high substrate concentration for the RuBisCO, which catalyzse the fixation of carbon dioxide and ribulose 1,5-bisphosphate into 3-phosphogylcerate. In the presence of oxygen (O<sub>2</sub>) the RuBisCO produces phosphoglycolate, a waste product which must be recycled by the cell. As carbon dioxide and oxygen competivly bind as substrates of the RubisCO, the accumulation of carbon dioxide by the carbonic anhydrase and the prevention of oxygen diffusion by the barrier of the carboxysome shell enables an efficient carbon dioxide fixation
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In nature there are plasmids which encode different proteins of the carboxysome in bacteria. One such plasmid is <i>pHnCBS1D</i> (Link dazu) which was found in <i>Halothiobacillus neapolitanis</i> (Link dazu) (REFERENZ). Different parts of this plasmid were used for the production of BioBricks (alle erzeugten BBs hier nennen + verlinken). 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 <i>csoS1A</i> and <i>gfp</i> (BioBrickNummer von unserem GFP + Link) as indicator of correct protein folding. A concentrated subcellular localisation ot the fluorescence shows the position of carboxysoms. This reporter function of GFP was identified and used before (REFERENZEN).
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        <h6>Shell protein folding</h6>
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The expression of <i>csoS1A:gfp</i> 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 <i>sap</i> and the <i>cso</i> coding sequences yields green fluorescence. This fluorescence was quantified by photometric measurements (Link auf Methode und auf Ergebnisse). The signal intensity of the <i>E. coli</i> KRX wildtype (negative control) was comparted to cells carrying either a plasmid with or without <i>sap</i>. <i>E. coli</i> 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.
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[Tabelle / Grafik mit Ergebnissen vom GloMax]
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         <h6>Carboxysome assembly</h6>
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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.
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This assembly was possible without the expression of <i>csoS1D</i> another coding sequence which is located on <i>pHnCBS1D</i>. The resulting protein is probably responsible for the pore size in the carboxysome envelope (REFERENZ). Nevertheless it seems that it has no essentiell function.
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[mikroskopische Aufnahmen => ggf. Evolution der Mikroskope]
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        <h6>Summary and outlook</h6>
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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 <i>E.coli</i>. One possible application beside the carbon dioxide fixation could be the fixation of atmospheric nitrogen which is essentiell for the growth of plants.
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Revision as of 21:47, 15 October 2014



CO2 fixation

Abstract

The expression of 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 (Link dazu) which was found in Halothiobacillus neapolitanis (Link dazu) (REFERENZ). Different parts of this plasmid were used for the production of BioBricks (alle erzeugten BBs hier nennen + verlinken). 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 (BioBrickNummer von unserem GFP + Link) as indicator of correct protein folding. A concentrated subcellular localisation ot the fluorescence shows the position of carboxysoms. This reporter function of GFP was identified and used before (REFERENZEN).

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.

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