Difference between revisions of "Part:BBa K1499500"
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After ligating this construct together and into the BioBrick backbone, we transformed it into the NEB 5-alpha strain of E. coli. The colonies that fluoresced were most likely to have been successfully transformed, and thus these were sent off for sequencing. The sequencing data showed that our construct was correct (see below), so we were able to submit for sequencing). | After ligating this construct together and into the BioBrick backbone, we transformed it into the NEB 5-alpha strain of E. coli. The colonies that fluoresced were most likely to have been successfully transformed, and thus these were sent off for sequencing. The sequencing data showed that our construct was correct (see below), so we were able to submit for sequencing). | ||
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Although our sequencing data was good, we realized that we had not induced the lac-repressible promoter, and thus the colonies should not have been fluorescing yet. After doing more research, we realized that | Although our sequencing data was good, we realized that we had not induced the lac-repressible promoter, and thus the colonies should not have been fluorescing yet. After doing more research, we realized that | ||
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Revision as of 20:53, 14 September 2014
quorum sensing machinery that activates GFP expression
This part is the combination of two parts, BBa_I13202 and BBa_T9002. The first part (BBa_I13202) produces a luxI enzyme which produces an AHL (3OC6HSL). This part is under the control of a lac repressible promoter. The second part (BBa_T9002) produces luxR, which combines with the AHL to activate the luxPR promoter that controls expression of GFP (see image below - created by Cambridge 2010 iGem Team, http://2010.igem.org/Team:Cambridge/ProjectBioluminescence/Luciferase/WikiGeneticsLuxCDABE).
Our goal in building this construct is to create a time delay. Since the production of AHL is under the control of a lac-repressible promoter, the cascade should not begin until the promoter is induced. Additionally, once the promoter is activated, time is required for the AHL and luxR molecules to build up and interact with one another. Only after these molecules are present in large enough quantities to interact and bind to the luxPR promoter will GFP be expressed. Thus by replacing GFP with any gene of interest, this construct can be used to create a time delay for the onset of gene expression. For more information on the specifics of the time delay, see the "experience" page.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1815
Illegal BsaI.rc site found at 2543