Difference between revisions of "Part:BBa K389014:Design"
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===Design Notes=== | ===Design Notes=== | ||
* The ''virA'' gene is under the control of a medium strong constitutive promoter so there is enough VirA receptor expressed but not too much so the cell suffers from the expression (VirA is a membrane protein) | * The ''virA'' gene is under the control of a medium strong constitutive promoter so there is enough VirA receptor expressed but not too much so the cell suffers from the expression (VirA is a membrane protein) | ||
| + | * The ''virG'' gene is mutated so it works without an ''rpoA'' subunit from ''Agrobacterium tumefaciens'' (YC Jung ''et al.'', 2004) | ||
* double terminator (forward) to keep expression of kanamycin resistance by the constitutive promoter low | * double terminator (forward) to keep expression of kanamycin resistance by the constitutive promoter low | ||
** double terminator <partinfo>B0017</partinfo> instead of <partinfo>B0015</partinfo> because of problems mentioned with <partinfo>B0012</partinfo> | ** double terminator <partinfo>B0017</partinfo> instead of <partinfo>B0015</partinfo> because of problems mentioned with <partinfo>B0012</partinfo> | ||
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===Source=== | ===Source=== | ||
| − | ''A. tumefaciens'' C58 | + | * ''virA'' from ''A. tumefaciens'' C58 (<partinfo>K389001</partinfo>) |
| + | * ''virG'' gene synthesized by Mr. Gene (<partinfo>K389002</partinfo>) | ||
| + | * ''vir'' promoter from ''A. tumefaciens'' C58 (<partinfo>K389003</partinfo>) | ||
| + | * ''kanR'' gene made from BioBrick <partinfo>P1003</partinfo> | ||
| + | * constitutive promoter, RBS and double terminator from parts.igem (<partinfo>J23110</partinfo>, <partinfo>B0034</partinfo>, <partinfo>B0017</partinfo>) | ||
===References=== | ===References=== | ||
| + | YC Jung ''et al.'' (2004) Mutants of ''Agrobacterium tumefaciens virG'' Gene That Activate Transcription of ''vir'' Promoter in ''Escherichia coli'', ''Current Microbiol'' 49:334-340. | ||
Latest revision as of 15:14, 16 October 2010
VirA/G screening system test
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 647
Illegal NheI site found at 2581
Illegal NheI site found at 2604 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1632
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1768
Design Notes
- The virA gene is under the control of a medium strong constitutive promoter so there is enough VirA receptor expressed but not too much so the cell suffers from the expression (VirA is a membrane protein)
- The virG gene is mutated so it works without an rpoA subunit from Agrobacterium tumefaciens (YC Jung et al., 2004)
- double terminator (forward) to keep expression of kanamycin resistance by the constitutive promoter low
- only for testing the screening principle of mutated virA screening
Source
- virA from A. tumefaciens C58 (BBa_K389001)
- virG gene synthesized by Mr. Gene (BBa_K389002)
- vir promoter from A. tumefaciens C58 (BBa_K389003)
- kanR gene made from BioBrick BBa_P1003
- constitutive promoter, RBS and double terminator from parts.igem (BBa_J23110, BBa_B0034, BBa_B0017)
References
YC Jung et al. (2004) Mutants of Agrobacterium tumefaciens virG Gene That Activate Transcription of vir Promoter in Escherichia coli, Current Microbiol 49:334-340.