Difference between revisions of "Part:BBa K4229049"

Revision as of 11:47, 12 October 2022

"Full wiffelball" with 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, the T1 protein is tagged with a Snoop and SpyTag. For the full wiffleball with tags please refer to: BBa_K4229048. The minimal wiffleball without tags: BBa_K4229046. The minimal wiffleball with the tags: BBa_K4229047.

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]

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, culture, induction and expression conditions are highly sensitive for microcompartments since they tend to form insoluble aggregates. We also fractioned the cell lysate to observe the solubility of the wiffleballs. All experiments were repeated a total of three times, with the exception of the Snoop-catching experiments. These were just performed two times.

[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/Snp 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/Snp-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 as that of the T 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

Sequence and Features