Designed by: Nikita Edel   Group: iGEM22_Freiburg   (2022-09-29)

Assembly of "minimal wiffelball" under regulation of lambdapLhybrid promotor and LacI promotor

This Biobrick shows the minimal wiffleball without any tags. Here, the two minimal wiffleballs will be compared. For the minimal wiffle with tags please refer to BBa_K4229047. The full wiffleball without tags: BBa_K4229048. The full wiffleball with tags: BBa_K4229049. The BMC proteins form wiffleballs, synthetic scaffolds that, when tagged with Snoop and Spy tags, can recruit enzymes, proteins or molecules tag with the analogue catcher.

We used fluorescent microscopy to monitor the localization of fluorescent proteins (FPs) fused to the Spy and Snoop(Snp)-Catchers when co-expressed with the proteins required to form the full and the minimal wiffleballs, with the T1 protein being fused to the Spy and SnpTags. Specifically, the SpyCatcher is fused to mVenus2 and the SnpCatcher to mTurquoise2 (Figure 2). We expected that the uptake of the FPs into the compartments should alter the fluorescence distribution in the cells: instead of cytoplasmic fluorescent signal, we would expect the formation of fluorescent foci To confirm the formation of the peptide bond linking the FP to the T1 protein, we performed Western Blotting. If the peptide bond is formed, the bands corresponding to the FP and the T1 protein are expected to run higher in the gel because of the higher molecular weight. An anti-His antibody was used against the His-tag of the T1 protein and an antibody against the beta-subunit of the RNA Polymerase was used a loading control.

We used the same BL21 cells for the microscopy and the Western Blot. After an induction test, we decided to use 100 µM IPTG to induce the proteins of the wiffleball and 50 ng/µl doxycycline to induce expression of the FP (mVenus2 or mTurquoise2). The bacteria, derived from overnight cultures and a prior incubation at 30°C, 200rpm, were induced at OD 600 = 0.6-0.7. Samples were taken after an incubation time of 24h with 200rpm at 18°C. We found this induction condition in the literature and at first decided to stick to these conditions since it has been often claimed that microcompartments are hard to express and often form insoluble aggregates. We also fractionated the cell lysate to observe the solubility of the BMCs. All experiments were repeated 3 times, with the exception of the experiments with the SnpCatcher, 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/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 fewer 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 3B).

[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 3 and 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 (Figure 4). 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 minimal wiffleball with and w/o tags (pHT1) + mVenus2

[Fig.3]Western Blot comparison of the BMC minimal wiffleball with and w/o tags (pHT1) + mVenus2

Sequence and Features

Assembly Compatibility:
  • 10
    Illegal EcoRI site found at 444
  • 12
    Illegal EcoRI site found at 444
    Illegal NotI site found at 1323
  • 21
    Illegal EcoRI site found at 444
    Illegal BglII site found at 453
  • 23
    Illegal EcoRI site found at 444
  • 25
    Illegal EcoRI site found at 444
    Illegal NgoMIV site found at 335
    Illegal NgoMIV site found at 842
    Illegal AgeI site found at 1127
    Illegal AgeI site found at 1235
  • 1000