Introduction

Naturally occuring, 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). We used different parts of this plasmid for the production of BioBricks. These BioBricks (BBa_K1465202-BBa_K1465223) were used for the construction of a synthetic carboxysome-encoding plasmid. It consists up of the shell proteins CsoS1ABC and CsoS4AB and the shell associated protein CsoS2. Thereby, the proteins should form an anaerobic microcompartment. This bottom up approach allows verifying the essentiality of the different components. We used a translational fusion of one of the shell proteins CsoS1A and GFP (BBa_K1465222) as an indicator of correct protein folding. A concentrated subcellular localisation of the gfp-fluorescence shows the positions of carboxysomes within the cells. This reporter function of GFP was identified and used for this purpose 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 (CsoS1A) with the CDS of the green fluorescent protein as an indicator of correct protein folding. The observed fluorescence in the carboxysome-expressing E. coli cells is concentrated at different points of the cell and indicates the presence of functional carboxysomes.

Shell protein folding

With our GFP fusion we wanted to analyze the protein folding of our constructed microcompartment, the carboxysome. On the one hand we had a GFP fusion with the shell proteins. On the other hand we analyzed the GFP fusion of the microcompartment with the additional shell associated protein encoded by csoS2 upstream of the shell proteins on the plasmid. The exact function of this protein is still unknown (Yeates et al., 2008).

Figure 1: Fluorescence measurement of E. coli KRX wild type (negative control) and E. coli KRX carrying one of the three different plasmids pSB1C3_p_Tet_gfp (positive control), 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 successful assembly of the carboxysome we analyzed the subcellular localization 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 wild type KRX (negative control), (B) Expression of E. coli KRX with plasmid pSB1C3_p_Tet_gfp (positive control), (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.

Figure 3: Fluorescence pictures taken through a structured illumination microscope. (A) E. coli wild type KRX (negative controls), (B) Expression of  coli KRX with plasmid pSB1C3_pTet_gfp (positive control), (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.

Figure 4: Fluorescence picture of a single E. coli carrying the pSB1C3_T7_csoS2_csoS4AB_csoS1CA_gfp_csoS1B plasmid and forming a filament. Picture taken through a structured illumination microscope.

Figure 5: Fluorescence picture of a single E. coli carrying the pSB1C3_T7_csoS2_csoS4AB_csoS1CA_gfp_csoS1B plasmid. Picture taken through a structured illumination microscope.

Summary and outlook

We report the construction of a synthetic plasmid encoding a functional carboxysome. Despite the fact that it has not shown the fixation of carbon dioxide in this compartment yet, we would like to emphasize the construction of a useful microcompartment. The functional carboxysome could be used for the carbon dioxide fixation. Therefore, three enzymes need 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 be cloned 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.
Different microscopy approaches showed a distinct fluorescence spot inside the cell. In contradiction the positive control showed a fluorescence throughout the whole cell. By doing this microscopy pictures we could identify fully assembled carboxysomes as well as filaments which result from the overexpression through the T7 promoter as described before.
The complete carboxysome could be used for anaerobic reactions in aerobic cultures of E. coli. One possible application beside the carbon dioxide fixation could be the fixation of atmospheric nitrogen which is essential for the growth of plants. With a size of approximately ~100-300 nm, all desired proteins that are smaller can be brought into our microcompartment for further experiments.

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.
• 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.
• Yeates et al., 2011. Protein-based organelles in bacteria: carboxysomes and related microcompartments. Nature Reviews Microbiology, vol. 6, pp. 681-691