Difference between revisions of "Part:BBa K917010"

Latest revision as of 12:33, 30 September 2012

Kanamycin resistance+lac promoter+RFP+sacB selection-counterselection cassette

This is an improved version of parts BBa_K322922 and K322923, which consisted of the Bacillus subtlis 168 levansucrase gene, sacB, a widely used counterselectable marker which is lethal when expressed in E. coli and other Gram negative bacteria in the presence of high sucrose concentrations, together with a kanamycin (BBa_P1003) or chloramphenicol (BBa_K322210) resistance marker. In this version, the lac promoter and Red Fluorescent Protein (RFP) from BBa_J04450 (but without the terminator) have been added. The provides a controllable promoter for sacB and also a marker gene. This is valuable as sacB counter-selection is notoriously prone to escape due to loss-of-function mutations in sacB. By including RFP, we can distinguish true loss of the cassette (giving white colonies) from loss of sacB function (which should still give red colonies). This will make our experiments much easier to interpret.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1756
Illegal AgeI site found at 1868
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K917010 short</partinfo>
-The Red Fluorescent Protein (RFP) and SacB, a Bacillus subtilis levansucrase created by Edinburgh team 2010 have been placed under the control of the lac promoter in addition to kanamycin resistance gene. Function is to create a selection-counterselection cassette.
+This is an improved version of parts '''BBa_K322922''' and '''K322923''', which consisted of the ''Bacillus subtlis'' 168 levansucrase gene, ''sacB'', a widely used counterselectable marker which is lethal when expressed in ''E. coli'' and other Gram negative bacteria in the presence of high sucrose concentrations, together with a kanamycin (BBa_P1003) or chloramphenicol (BBa_K322210) resistance marker. In this version, the ''lac'' promoter and Red Fluorescent Protein (RFP) from BBa_J04450 (but without the terminator) have been added. The provides a controllable promoter for ''sacB'' and also a marker gene. This is valuable as ''sacB'' counter-selection is notoriously prone to escape due to loss-of-function mutations in ''sacB''. By including RFP, we can distinguish true loss of the cassette (giving white colonies) from loss of ''sacB'' function (which should still give red colonies). This will make our experiments much easier to interpret.
 <!-- Add more about the biology of this part here