Part:BBa_K4229044

H,T1,T2,T3 assemble the "full wiffelball"

This biobrick is a combination of the biobricks BBa_K4229035, BBa_K4229036 and BBa_K4229037, which together assemble to form the full wiffleball structure. In this biobrick, the T1 protein is not tagged (BBa_K4229030). Here, we explain what bacterial microcompartments are and how the wiffleballs are built. For experimental data look at the registry site of biobrick: BBa_K4229048.

Bacterial microcompartments (BMCs) are self-organising organelles with a selectively permeable protein shell. All BMCs consist of three conserved families of proteins: BMC-H (forming hexamers), and BMC-T (pseudohexamers) both with pores of different sizes in the middle and BMC-P (pentamers) [1][2]. Small molecules can enter the lumen of BMCs via the pores found within the BMC-H shell proteins (which vary in size from 4 - 7Å in diameter) or the larger pores (~12 - 14 Å in diameter) formed by BMC-T trimers which can have an open or closed conformation [3][4]. For our project, we used the recently published synthetic BMCs from Kirst et al [2], which are based on the shell system from the myxobacterium Haliangium ochraceum (HO-shell) (Figure 3A). The HO-shell is able to assemble without containing any cargo molecule inside [2][5] and is built by the shell proteins BMC-H, BMC-P and three BMC-T proteins (single-layer T1 and double-layer T2 and T3). The synthetic BMC shell, designed by the Kerfeld lab can form without the presence of the BMC-P proteins [6][7]. Without the pentamers, there are pores left that allow molecules to diffuse in/out of the lumen of the BMC. This form of synthetic BMC is called full wiffleball. An even more simplified shell (minimal wiffleball) was designed to consist of only two shell proteins, BMC-H and BMC-T1.

Synthetic BMCs serve as autonomous metabolic modules, which are decoupled from the regulatory mechanisms of the cell and are only connected to the metabolism of the cell via the engineered protein envelope [2]

A: Showing the original HO-Shell found in Haliangium ochraceum. B: showing the full wiffleball made with H-/T1-/T2-/T3-protein. C: minimal wiffleball made just our of the H- and T1-protein.

[1] C. A. Kerfeld, C. Aussignargues, J. Zarzycki, F. Cai, and M. Sutter, “Bacterial microcompartments,” Nat. Rev. Microbiol., vol. 16, no. 5, pp. 277–290, 2018, doi: 10.1038/nrmicro.2018.10.

[2] H. Kirst, B. H. Ferlez, S. N. Lindner, C. A. R. Cotton, A. Bar-Even, and C. A. Kerfeld, “Toward a glycyl radical enzyme containing synthetic bacterial microcompartment to produce pyruvate from formate and acetate,” Proc. Natl. Acad. Sci. U. S. A., vol. 119, no. 8, pp. 1–10, 2022, doi: 10.1073/pnas.2116871119.

[3] H. Kirst, B. H. Ferlez, S. N. Lindner, C. A. R. Cotton, A. Bar-Even, and C. A. Kerfeld, “Toward a glycyl radical enzyme containing synthetic bacterial microcompartment to produce pyruvate from formate and acetate,” Proc. Natl. Acad. Sci. U. S. A., vol. 119, no. 8, pp. 1–10, 2022, doi: 10.1073/pnas.2116871119.

[4] M. J. Lee, D. J. Palmer, and M. J. Warren, “Biotechnological Advances in Bacterial Microcompartment Technology,” Trends Biotechnol., vol. 37, no. 3, pp. 325–336, 2019, doi: 10.1016/j.tibtech.2018.08.006.

[5] J. K. Lassila, S. L. Bernstein, J. N. Kinney, S. D. Axen, and C. A. Kerfeld, “Assembly of robust bacterial microcompartment shells using building blocks from an organelle of unknown function,” J. Mol. Biol., vol. 426, no. 11, pp. 2217–2228, 2014, doi: 10.1016/j.jmb.2014.02.025.

[6] H. Kirst and C. A. Kerfeld, “Bacterial microcompartments: Catalysis-enhancing metabolic modules for next generation metabolic and biomedical engineering,” BMC Biol., vol. 17, no. 1, pp. 1–11, 2019, doi: 10.1186/s12915-019-0691-z.

[7] A. Hagen, M. Sutter, N. Sloan, and C. A. Kerfeld, “Programmed loading and rapid purification of engineered bacterial microcompartment shells,” Nat. Commun., vol. 9, no. 1, pp. 1–10, 2018, doi: 10.1038/s41467-018-05162-z.

The plasmid containing this biobrick was kindly send to us by the Kerfeld group see [2].

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1061
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1061
  Illegal NotI site found at 865
  Illegal NotI site found at 2242
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1061
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1061
  Illegal NgoMIV site found at 384
  Illegal NgoMIV site found at 1862
  Illegal NgoMIV site found at 2218
  Illegal AgeI site found at 669
  Illegal AgeI site found at 777
  Illegal AgeI site found at 1364
  
• 1000
  COMPATIBLE WITH RFC[1000]