Part:BBa_K4229046
Assembly of "minimal wiffelball" under regulation of lambdapLhybrid promotor and LacI promotor
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.
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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