Part:BBa_K4229046
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.
We expressed tagged mVenus2 either alone or co-expressed with the wiffleballs, which were either tagged or not tagged with the Spy/Snoop-tag at the T1 protein. The results gave us important insights: samples with tagless wiffleballs show an evenly distributed fluorescence [Fig.2:A]. On the other hand, the T1 with tags resulted in fluorescent foci in some cells standing out of the fluorescent background [Fig.2B arrows]. The foci in the full wiffleball were always found at one or both poles of the cells. The minimal wiffleball had fewer and smaller foci, which were also localized in other areas of the bacteria. The foci were easier detectable in less fluorescent cells. Therefore, more foci could be hidden in cells with brighter fluorescence. BL21(DE3) showed in some induced cells a stretched phenotype [Fig.2:B].
By western blotting, the expression of the T1 could be proven by a 29kDa (T1 w/o tags) and a 37kDa (T1) band [Fig.3]. The absence of the Spy/Snoop-tag resulted in one single band. Expression of the tageed T1 protein led to a second band, shifted to around 80kDa. The molecular weight of mVenus2-SpyCatcher has the same size as the T1 with the tags (37kDa). Both in the full and minimal wiffleball the catching seems to be successful. A small fraction of the unbound T1 was found in the insoluble fraction of the cells each time, except when fused to mVenus2. This excludes the possibility that the nature of foci results from insoluble T1-mVenus2-aggregates. Most insoluble T1 was found in the pellet of the minimal wiffleball when mVenus2 was also expressed.
By western blotting, the expression of the T1 could be proven by a 29kDa (T1 w/o tags) and a 37kDa (T1) band [Fig.3]. The absence of the Spy/Snoop-tag resulted in one single band. Expression of the tageed T1 protein led to a second band, shifted to around 80kDa. The molecular weight of mVenus2-SpyCatcher has the same size as the T1 with the tags (37kDa). Both in the full and minimal wiffleball the catching seems to be successful. A small fraction of the unbound T1 was found in the insoluble fraction of the cells each time, except when fused to mVenus2. This excludes the possibility that the nature of foci results from insoluble T1-mVenus2-aggregates. Most insoluble T1 was found in the pellet of the minimal wiffleball when mVenus2 was also expressed.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 444
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 444
Illegal NotI site found at 1323 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 444
Illegal BglII site found at 453 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 444
- 25INCOMPATIBLE WITH RFC[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 - 1000COMPATIBLE WITH RFC[1000]
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