Difference between revisions of "Part:BBa K1669009"

 
 
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Bdhb fused with strong promotor, strong RBS and double terminator
 
Bdhb fused with strong promotor, strong RBS and double terminator
  
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===Usage and Biology===
 
===Usage and Biology===
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To investigate the activity of the construct we utilized SDS-PAGE and Western blot.
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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.
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https://static.igem.org/mediawiki/2015/thumb/5/56/SdsBdhB.jpeg/215px-SdsBdhB.jpeg
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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.
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https://static.igem.org/mediawiki/2015/thumb/5/51/MembranaBdhB.jpeg/200px-MembranaBdhB.jpeg
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Latest revision as of 22:40, 17 September 2015

BdhB with strong promotor, strong RBS and double terminator

Bdhb fused with strong promotor, strong RBS and double terminator


Usage and Biology

To investigate the activity of the construct we utilized SDS-PAGE and Western blot. 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.

215px-SdsBdhB.jpeg


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.

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1082