Difference between revisions of "Part:BBa K2066115"

Latest revision as of 03:27, 29 October 2016

Synthetic Enhancer Project: 2x TetO Binding Cassette (55as) + 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 2x TetO binding cassette within the spacer region between the enhancer and promoter. The part was characterized via insertion of an sfGFP reporter under the control of the sigma 54 promoter. The two binding sites allows for three discrete states of output: a repressed, intermediate, and 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
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 890
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 1878

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2066115 short</partinfo>
-This part takes the synthetic genetic circuit from Amit et. al. and puts it onto a BioBrick backone flanked by the UNS regions. Used for the synthetic enhancer project, this part consists of an enhancer region, that when bound to phosphorylated NRI protein, can bind to the promoter after the DNA loops to allow for these kinetics.  Once the looping and interaction between the enhancer and promoter happens, transcription of the NRI protein for positive feedback as well as the output flourescent reporter is initiated.  This part is from the synthetic enhancer genetic circuits created by Amit et. al where there is a tetR binding cassette with two TetO binding sites in the spacer region between the enhancer and the promoter.  The two binding sites allows for three discrete states of output: a repressed, intermediate, and unrepressed state. Small chemical induction with aTc binds to and inactivates available TetR repressor.  When a small amount of aTc is present, there isn't enough aTc to find TetR proteins and there is enough repressor available to allow for a constant filling of the two available TetO binding sites. When both TetO sites are bound, the DNA becomes less flexible and makings looping of the enhancer to the promoter extremely difficult, thus not allowing for very minimal transcription of the NRI and reporter proteins.  When more aTc is added to the system, more TetR is bound and inactivated and this creates an intermediate step where enough TetR is bound that at any point only one TetR is bound to the teto sites and this increases the rigidity of the DNA and allows for looping and activation and reporter output less frequently.  Finally, when aTc conentration is high, you reach a saturation point where almost all TEtR repressors are bound and inactivated and you get maximal amount of looping and thus maximal amount of NRI and reporter transcribed and translated.
+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 2x TetO binding cassette within the spacer region between the enhancer and promoter. The part was characterized via insertion of an sfGFP reporter under the control of the sigma 54 promoter. The two binding sites allows for three discrete states of output: a repressed, intermediate, and 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: