Difference between revisions of "Part:BBa K1669007"
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CtfA fused with strong promotor, strong RBS and double terminator. | CtfA 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. | ||
| + | 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/6/6c/PraviSDSctfA.jpeg/203px-PraviSDSctfA.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/7/7f/MembranaCtfB.jpeg/188px-MembranaCtfB.jpeg | ||
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Latest revision as of 22:30, 17 September 2015
CtfA with strong promotor, strong RBS and double terminator
CtfA 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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 709
Illegal AgeI site found at 236 - 1000COMPATIBLE WITH RFC[1000]