Difference between revisions of "Part:BBa K4229048"

Latest revision as of 02:18, 14 October 2022

"Full wiffelball" without tags, under regulation of lambdapLhybrid promotor and LacI promotor

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, no BMC protein is tagged with Snoop and SpyTag. 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.

Biology

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].

Experimental setup

The formation of the wiffleballs inside E. coli cells can be indirectly measured by the formation of fluorescent foci due to the recruitment into the shells of the fluorescent protein(s) mVenus2 and/or mTurquoise2, each respectively fused to the SpyCatcher and SnoopCatcher. We used the same induced BL21 cells for the microscopy and the Western blot. After an induction test, we decided to use 100µM IPTG for wiffleball induction and 50ng/µl doxycycline for the fluorescent protein expression (mVenus2 or mTurquoise2). The bacteria were grown in overnight cultures shaken at 30°C, 200rpm, induced at OD600= 0.6-0.7. Samples were taken after 24h of incubation at 200 rpm at 18°C. Conditions were based on literature research . In general, finding the right culture, induction and expression conditions for microcompartment formation is a delicate task since the proteins forming the microcompartments tend to form insoluble aggregates. We also fractionated the cell lysate to observe the solubility of the wiffleballs. All experiments were repeated a total of three times, with the exception of the experiments with mTurquoise2-SnoopCatcher, which were performed only twice.

[Fig.1]Principle of catching proteins via Spy/Snp-Catcher by T1 and their incorporation into the BMCs, illustrated as an example of incorporating mVenus2 in the full wiffleball

The expression of mVenus2 alone and together with the wiffleballs, in which the T1 protein lacked the Spy/Snoop tags, showed a homogeneous distribution of the fluorescence within the cells (Figure 1A; B). When the T1 protein had the tags, we observed the appearance of fluorescent foci in some cells (Figure 3B arrows). The foci in the cells expressing the full wiffleball were always found at one or both poles of the cells. Cells expressing the minimal wiffleball had less and smaller foci, which were also localized in other areas of the bacteria. The foci were more easily detectable in less fluorescent cells. Therefore, more foci could be hidden in cells with brighter fluorescence. Some BL21(DE3) cells became elongated when expressing the wiffleball (Figure 2B).

[Fig.2]Fluorescent microscopy of T1 catching the mVenus2, when the minimal or full wiffleball construct is expressed; A: Controls for the induction; B: T1 with and without the Spy/Snp tags; scalebar 5µm

We could detect the T1 protein (29 kDa w/o tags or 37 kDa with tags) on the Western Blot (Figure 4). The absence of the Spy/Snoop-tag resulted in one single band. When the tag was present, a second band corresponding to circa 80 kDa was observable. The molecular weight of mVenus2-SpyCatcher is the same of that of the T1 protein with the tags (37 kDa). We observed the formation of the peptide bond both with the full and the minimal wiffleball (Figure 4). Always a small fraction of the unbound T1 was found in the insoluble fraction of the cells, but not when fused to mVenus2, which excludes the possibility that the nature of foci results from insoluble T1-mVenus2 aggregates. Most insoluble T1 was found in the pellet of cells expressing the minimal wiffleball, when mVenus2 was also expressed.

[Fig.3]Western Blot comparison of the BMC full wiffleball with and w/o tags (pT1T2T3) + mVenus2

[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] M. G. Klein et al., “Identification and Structural Analysis of a Novel Carboxysome Shell Protein with Implications for Metabolite Transport,” J. Mol. Biol., vol. 392, no. 2, pp. 319–333, 2009, doi: 10.1016/j.jmb.2009.03.056.

[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 the full wiffleball was a kind gift by Cheryl Kerfeld. Sequence and Features