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Team:Bielefeld-CeBiTec/Results/CO2-fixation/Carboxysome
From 2014.igem.org
Module II - Carbon Dioxide (CO2) Fixation
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 neapolitanus (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 carboxysomes. This reporter function of GFP was identified and used before (Waldo et al., 1999; Hsu et al., 2009).
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.
Shell protein folding
With our GFP fusion we wanted to analyse the protein folding of our constructed microcompartiment, the carboxysome. On the one hand we had a GFP fusion only with the shell proteins and on the other hand we analysed a GFP fusion of the microcompartiment but with the additionally shell assosiated protein encoded by csoS2.
Figure 1: Fluorescence measurement of E. coli KRX wildtype and E. coli KRX carrying one of the three different plasmids pSB1C3_pTet_gfp, pSB1A2_T7_csoS4AB_csoS1CA_gfp_csoS1B and pSB1C3_T7_csoS2_csoS4AB_csoS1CA_gfp_csoS1B. The measurement has been done with the GloMax® Discover Multimode-Reader.
Carboxysome assembly
To show the successfull assembly of the carboxysome we analysed the subcellular localisation of the green fluorescent protein. Pictures taken through a fluorescence microscope are shown in figure 2.
Figure 2: Fluorescence pictures taken through a Leica DMI6000 microscope. (A) E. coli wildtype KRX, (B) Expression of E. coli KRX with plasmid pSB1C3_pTet_gfp, (C) Expression of E. coli KRX with plasmid pSB1A2_T7_csoS4AB_csoS1CA_gfp_csoS1B, (D) and (E) Expression of E. coli KRX with plasmid pSB1C3_T7_csoS2_csoS4AB_csoS1CA_gfp_csoS1B.
To get a higher resolution of the carboxysomes in the cells we also used a structured illumination microscope . The pictures are shown in figure 3.
Figure 3: Fluorescence pictures taken through a structured illumination microscope microscope. (A) E. coli wildtype KRX, (B) Expression of coli KRX with plasmid pSB1C3_pTet_gfp, (C) Expression of E. coli KRX with plasmid pSB1A2_T7_csoS4AB_csoS1CA_gfp_csoS1B, (D) and (E) Expression of E. coli KRX with plasmid pSB1C3_T7_csoS2_csoS4AB_csoS1CA_gfp_csoS1B. With this higher resolution we could identify the carboxysomes in the cells with the pSB1C3_T7_csoS2_csoS4AB_csoS1CA_gfp_csoS1B plasmid in one specific location of the cell (picture D and E). Most of the cells seem to have only one carboxysomes but there were also some that carried two. Additionally we could detect some cells forming a filament located at the border of the cell (figure 4). These filaments might be formed because of a high induction of the plasmid ( Bonacchi et al., 2012). As we used the strong T7 promoter this could be the reason for this occurrence.
Figure 3: Fluorescence picture of a single E. coli carrying the pSB1C3_T7_csoS2_csoS4AB_csoS1CA_gfp_csoS1B and forming a filament. Picture taken through a structured illumination microscope microscope.
The assembly of the carboxysome 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.
Carboxysome purification
Purification of recombinant proteins is one essential foundation of syntetic biology. For this reason, it was our first approach to purify the carboxysome from the chemolitoautotroph model organism Halothiobacillus neapolitanus, recombinant expressed in E. coli. After establishment of the methode, our constructed carboxysome should be purified with this methode. The work for purifying carboxysomes was nevertheless based on the plasmide pHnBCS1D, which is coding for the H. neapolitanus carboxysome. 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 4-7:
 Figure 5: Proteinexpression of pHnCBS1D induced with 0.5 mM IPTG |
 Figure 6: Proteinexpression of pHnCBS1D induced with 2 mM IPTG |
 Figure 7: Proteinexpression of pHnCBS1D induced with 5 mM IPTG |
As this SDS-PAGE shows the cellular proteins at different times points in the cultivation, you can recognize that 12 hours after induction there is a new band somewhat smaler than 55 kDa. Analytics via MALDI-TOF identified this band as the large subunit of RuBisCO (52.6 kDa) from Halothiobacillus neapolitanus. The analysis was done via tryptic digestion in silico and alignment of the identified peptides with the in silico peptides. Four identical peptides could be found and the sequence coverage (MS) was 8.9 %. When protein expression was induced with 0.5 mM IPTG, after 12 h increases a band with a size of propably 13 kDa. MALDI-TOF displayed that this is the small subunit of the RuBisCo (12.8 kDa). It was identified via in silico digest and alignment. Two peptides were found and the sequence coverage (MS) was 28.2. When IPTG was added to 2 or 5 mM, it seems like that the intensity of the band of the small subunit decreases in time of cultivation. It could be possible, that the small subunit is degrated, but inconsistent is the increase of the large subunit. Degradation of the small subunit is not described in the literature. The fact that RuBisCo composes nearly 70 % of the protein mass (by weight) in the carboxysome could explain why other proteins from the carboysome could not be found in this SDS-PAGE.
The purification of the carboxysome is carried out via several centrifugation steps. The last step of this methode is sucrose gradient centrifugation. This method did not work in our hands at all. Unfortunately, we were not able to correct the limiting step.
Two different methods for cell lysis were tryied, sonication and Frensh pressure cell, in their execution was ensured, that the mechanical stress for the cells could not destroy the microcompartiments. The following centrifugation steps seemed to be succesful, as the supernatants after centrifugation were analysed by SDS-PAGE, showing the expected result. After the first centrifugation for the seperation of cell debris, the supernatant carried the soluble proteins. After pelleting the carboxysomes, the supernatant of both centrifugation steps contained a low amount of protein.
The sample, which was layered on top of the sucrose gradient, had a slightly blue color, and it builds up a layer on the sucrose gradient, indicating that the gradient seems to be correctly formed. After ultracentrifugation there was no visble band of carboxysomes detectable. Unfortunately, we are not available of the equipment for scanning such gradients. SDS-PAGE of the fractionated gradient showed protein in every fraction, indicating that there was not enough concentrating force for concentration of the carboxysomes. A longer centrifugation periode resultet in a pellet, which had the same blue color than the sample, which was layered on the gradient. This suggest, that the carboxysomes were pelleted and the correct centrifugation time was not found.
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. The functional carboxysome could be used for the carbon dioxide fixation. Therefore three enzymes needs to be heterologous expressed in E. coli. We already tried to combine the carboxysome encoding genes and the three required enzymes on a single plasmid. The carbonic anhydrase (csoS3) should lay between the shell associated protein and the shell proteins. One RubisCO (BBa_K1465202) and one phosphoribulokinase (BBa_K1465201) encoding sequence should be integrated upstream of the carboxysomal operon.
The complete carboxysome 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.
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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.
