Difference between revisions of "Part:BBa K2066116"

Latest revision as of 03:16, 29 October 2016

Synthetic Enhancer Project: 3x TetO Binding Cassette (52s) + sfGFP on UNS

The synthetic enhancer, first characterized by Amit et. al. 2011, allows for a multistate transfer function by modulating the rigidity of the spacer region between the enhancer promoter region, thus affecting the DNA rigidity and hence the kinetic capability of the two components to loop together and initiate transcription. For more information on the mechanism, please refer to: http://2016.igem.org/Team:William_and_Mary/Synthetic_Enhancer.

This synthetic enhancer part consists of a 3x TetO binding cassette within the spacer region between the enhancer and promoter. The part was characterized using an sfGFP reporter. The three binding sites allow for four discrete states of output: a repressed, two intermediate, and an unrepressed state. Depending on the steady state of TetR, the amount of bound TetR varies and thus affects the rigidity of the spacer region as well as the kinetics of the looping between the enhancer and promoter, which allows for the initiation of transcription. This part has to be transformed in conjugation with the pACT Tet plasmid (Bba_K2066037), which controls the expression of TetR and NRII2302, a kinase that activates the NRI product (which binds and primes the upstream enhancer region).

Source: The enhancer, tet cassette, glnAp2 synthetic promoter, and NRI coding region sequences were derived from Amit, R., Garcia, H. G., Phillips, R. & Fraser, S. E. Building enhancers from the ground up: a synthetic biology approach. Cell146, 105–118 (2011). The sfGFP flourescent reporter design is inspired by C. Lou, B. Stanton, Y.-J. Chen, B. Munsky, C. A. Voigt, Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nat. Biotechnol. 30, 1137 (2012). doi:10.1038/nbt.2401 pmid:23034349. The UNS sequences at the ends of the insert are derived from Torella, J. P., Boehm, C. R., Lienert, F., Chen, J. H., Way, J. C., & Silver, P. A. (2013). Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic acids research, gkt860. A huge thanks to all the researchers involved in its original creation!

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 111
Illegal NheI site found at 206
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 171
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 951
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 1939

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2066116 short</partinfo>
-This part takes the synthetic enhancer circuit with a three TetO binding cassette and puts it onto a single BioBrick backbone flanked by the UNS regions. It includes a sfGFP reporter coding sequence downstream of the sigma 54 promoter. This part allows for a four step output response due to the three TetO binding cassette in the spacer region between the enhancer and promoter.The number of filled TetO sites influences the rigidity and the thermodynamics of the looping.
+The synthetic enhancer, first characterized by Amit et. al. 2011, allows for a multistate transfer function by modulating the rigidity of the spacer region between the enhancer promoter region, thus affecting the DNA rigidity and hence the kinetic capability of the two components to loop together and initiate transcription. For more information on the mechanism, please refer to: http://2016.igem.org/Team:William_and_Mary/Synthetic_Enhancer.
-This part should be transformed with the helper plasmid Bba_K2066037 to get a consititutive expression of TetR repressor and NRII2302 (which is a kinase and phosphorylates NRI to activate it and allow it to bind to the enhancer).
+This synthetic enhancer part consists of a 3x TetO binding cassette within the spacer region between the enhancer and promoter. The part was characterized using an sfGFP reporter. The three binding sites allow for four discrete states of output: a repressed, two intermediate, and an unrepressed state. Depending on the steady state of TetR, the amount of bound TetR varies and thus affects the rigidity of the spacer region as well as the kinetics of the looping between the enhancer and promoter, which allows for the initiation of transcription. This part has to be transformed in conjugation with the pACT Tet plasmid (Bba_K2066037), which controls the expression of TetR and NRII2302, a kinase that activates the NRI product (which binds and primes the upstream enhancer region).
 Source: