Difference between revisions of "Part:BBa K4229046"

Revision as of 11:29, 12 October 2022

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

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].

[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

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.

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

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.

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

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 444
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 444
Illegal NotI site found at 1323
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 444
Illegal BglII site found at 453
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 444
25
INCOMPATIBLE 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
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4229046 short</partinfo>
-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.
+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.
-The BMC proteins form wiffleballs, synthetic scaffolds that, when tagged with Snoop and Spy tags, can recruit enzymes, proteins or molecules tag with the analog catcher. We used fluorescent microscopy to monitor the catching of fluorescent proteins linked with the Spy and Snoop-catcher into the wiffleballs. The Spy-Catcher is fused to the fluorescent protein mVenus2 and the Snoop-Catcher to mTurquoise2 [Fig.1]. We expected that the uptake of the fluorescent proteins into the compartments should alter the fluorescence distribution in the cells, as most of the fluorescent protein is expected to be recruited into the microcompartment.
+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.
-Complementing our microscopy, we conducted western blots to assess the amount of caught protein bound to the wiffleball subunit BMC T1. Successful catching results in a shift of the bands upwards, due to the change in their molecular weight. For the detection of T1, an anti-His antibody was used against the His-tag of the T1. Furthermore, an antibody against the beta-subunit of the <i>E. coli</i> RNA Polymerase was used as a loading control. Both primary antibodies were detected with an anti-mouse-horseradish peroxidase (HRP) conjugate, which was detected with ECL-solution
+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
-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.
+induce expression of the FP (mVenus2 or mTurquoise2). The bacteria, derived from overnight
-All experiments have repeated a total of three times, with the exception of the Snoop-catching experiments. These were just performed two times.
+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.
 [[File:ReporterCatched.jpg|800px|thumb|left|[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]]