Intermediate

Part:BBa_K608002

Designed by: Julia Mller   Group: iGEM11_Freiburg   (2011-09-15)

strong Promoter and strong RBS

You can insert your gene of interest behind this part.
If you want a functional protein you need a promotor and a ribosome binding site for proper gene expression.


Usage and Biology

Slovenia HS team characterized this part in 2015.

The part was fused with our own BioBricks containing a coding region and a double terminator. The parts used were K1669002, which contains CtfA with His-tag, K1669005, which contains CtfB with His-tag, and K1669006, which contains BdhB with His-tag. To evaluate the activity of the promoter and RBS, proteins were expressed.

Escherichia coli DH5α bacteria were transformed with the expression plasmids and grown in shaker flasks at 37 °C in LBC medium overnight. 1 ml samples were taken for analysis. Cells were collected by centrifugation at 6000g for 10 min and resuspended in 100 µl of H20. 10 µl samples were taken and 5 µl of SDS-PAGE loading buffer was added. Samples were cooked at 100 °C for 10 min and resolved on 12% SDS-PAGE gel.

203px-PraviSDSctfA.jpeg 237px-SdsCtfB.jpeg 215px-SdsBdhB.jpeg


The proteins were successfully expressed and were entirely in the insoluble fraction, mostly likely due to the strength of the promoter and RBS. The procedure for checking expression was then repeated with bacteria grown at 20 °C and 16 °C, at which point some protein was observed in the soluble fraction as well.

Proteins were detected using Western blot as well. 12% SDS-PAGE gels were electroblotted for 1, 5 hours at 200 V, blocked in non-fat dry milk overnight and incubated the next day in two steps. A dilute solution of primary antibody was incubated with the membrane under gentle agitation for an hour. Then a dilute solution was incubated with the membrane under gentle agitation for an hour as well. The hydrogen peroxide method was for visualization.

188px-MembranaCtfB.jpeg 200px-MembranaCtfA%28vresniciB%29.jpeg 200px-MembranaBdhB.jpeg

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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