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

minimal wiffleball with tags under regulation of lambdapLhybrid promoter and LacI promoter

This Biobrick shows the minimal wiffleball without any of the tags. Here the two minimal wiffleballs will be compared. For the minimalwiffle without tags search: BBa_K4229046. The full wiffleball withouttags: BBa_K4229048. The full wiffleball with the tags: BBa_K4229049. We used fluorescent microscopy to monitor the uptake 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. The microscopy was done with a Zeiss Axiovert with Colibri-LEDs. 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 of 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 an antibody against the beta-subunit of the E. coli RNAPolymerase was used 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 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

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

Sequence and Features

Assembly Compatibility:
  • 10
    Illegal EcoRI site found at 444
  • 12
    Illegal EcoRI site found at 444
    Illegal NotI site found at 1515
  • 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 1319
    Illegal AgeI site found at 1427
  • 1000