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 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_p_Tet_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.

The fluorescence shown in figure 1 was quantified by photometric measurements. The signal intensity of the E. coli KRX wildtype (negative control) is compared to cells carrying a plasmid without csoS2 (BBa_K1465222). E. coli KRX cells with pSB1C3-p_Tet_gfp (BBa_I13522) serve as a positive control and lead to the highest fluorescence signal. The expression of csoS1A_gfp with all other known essentiell shell proteins (BBa_K1465222) does not yield any fluorescence. This indicates that the presence of the shell assosiated protein is needed for the correct folding of the shell proteins and the assembly of the whole carboysome. The combined expression of csoS2 and the coding sequences of the shell proteins csoS1CAB and csoS4AB (BBa_K1465223) yields green fluorescence. From these results we deduce that the correct folding of the shell proteins is dependent of the presence of the shell assosiated protein CsoS2.

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_p_Tet_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.

The negative control with the wildytpe of E. coli in picture A does not yield any fluorescence just as well as the sample with the plasmid pSB1A2_T7_csoS4AB_csoS1CA_gfp_csoS1B in picture C so we can see that the carboxysome cannot be folded correctly without the shell associated protein CsoS2. In picture B, the sample with the positive control, the whole cells cause fluorescence. The E. colis carrying the whole carboxysome plasmid with the GFP fusion are shown in picture D and E. As expected not the whole cells are fluorescing but only a small part of the cells. We assume that the concentrated green fluorescence results from GFP molecules which are fused to shell proteins of a successfully assembled carboxysome.
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.

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

• Cannon, Shively 1983. Characterisation of a Homogenous Preparation of Carboxysomes from Thiobacillus neapolitanus. Archive of Microbiology, vol. 134, pp. 52-59
• 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.
• 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.
• 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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