Difference between revisions of "Part:BBa K218006:Design"
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===Design Notes=== | ===Design Notes=== | ||
| − | The LuxPQ and LuxOU operons each have an internal RBS and terminator. Additional terminators were cloned downstream of each operon to ensure insulation. | + | The LuxPQ and LuxOU operons each have an internal RBS and terminator. Additional terminators were cloned downstream of each operon to ensure insulation. The TetR promoter (BBa_R0040) was cloned upstream of the LuxOU operon to allow for constitutive expression of these cytoplasmic proteins. No promoter has been cloned upstream of LuxPQ because the proteins encoded by this operon are found in the periplasm, an area of limited space. With this in mind, expression levels must be under exquisite control so the cell is not overloaded with the presence of LuxPQ. This control comes with designing a library of 256 different ∆σ70 promoters that will alter expression levels of LuxPQ. These promoters are flanked with BamHI and XhoI restriction sites and will be cloned into the pCS26 vector (Bjarnason et al., 2003), upstream of the cloning site for this signalling circuit. |
===Source=== | ===Source=== | ||
Revision as of 01:08, 22 October 2009
LuxPQ followed by terminator and LuxOU generator
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2209
Illegal BglII site found at 2427 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3509
Illegal BsaI.rc site found at 4886
Illegal SapI.rc site found at 2177
Illegal SapI.rc site found at 4848
Design Notes
The LuxPQ and LuxOU operons each have an internal RBS and terminator. Additional terminators were cloned downstream of each operon to ensure insulation. The TetR promoter (BBa_R0040) was cloned upstream of the LuxOU operon to allow for constitutive expression of these cytoplasmic proteins. No promoter has been cloned upstream of LuxPQ because the proteins encoded by this operon are found in the periplasm, an area of limited space. With this in mind, expression levels must be under exquisite control so the cell is not overloaded with the presence of LuxPQ. This control comes with designing a library of 256 different ∆σ70 promoters that will alter expression levels of LuxPQ. These promoters are flanked with BamHI and XhoI restriction sites and will be cloned into the pCS26 vector (Bjarnason et al., 2003), upstream of the cloning site for this signalling circuit.
Source
LuxPQ - GenBank Accession: NC_009784 Vibrio harveyi ATCC BAA-1116 chromosome II, REGION: 579654..580751.
LuxOU - GenBank Accession# L26221 LuxO Protein ID# AAD12736.1 LuxU Protein ID# AAD12737.2
B0015 - Registry of Standard Biological Parts
R0040 - Registry of Standard Biological Parts
References
Jeremy A. Freeman and Bonnie L. Bassler. Sequence and function of LuxU: a two-component phosphorelay protein that regulates quorum sensing in Vibrio harveyi. 1999. Journal of Bacteriology. Feb 1999, p. 899-906.
Neiditch MB, Federle MJ, Pompeani AJ, Kelly RC, Swem DL, Jeffrey PD, Bassler BL, Hughson FM. Ligand-induced asymmetry in histidine sensor kinase complex regulates quorum sensing. Cell. 2006 Sep 22;126(6):1095-108.