Difference between revisions of "Part:BBa K2066114"

Latest revision as of 02:07, 29 October 2016

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

This part takes the synthetic enhancer circuit with a three TetO binding cassette as well as the promoter and coding region of NRII from the helper plasmid of Amit et. al. 2011 circuits and puts it onto a single BioBrick backbone flanked by the UNS regions. 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. Combining the NRII and synthetic enhancer reduces metabolic strain, decouples the system from LacI/IPTG dependence, and allows for an ease of cloning due to the UNS sequences. The number of filled TetO sites influences the rigidity and the thermodynamics of the looping.

This part should be transformed with Bba_K2066022 to get constitutive expression of TetR repressor and allow for the circuit to output a 4 step multimodal response.

Source: The enhancer, tet cassette, glnAp2 synthetic promoter, NRI coding region, and mCherry 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 NRII2302 coding region and the promoter that it is controlled by is derived from the helper plasmid pACT tet from Amit et. al 2011. 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
Illegal NotI site found at 3773
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
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K2066114 short</partinfo>
-This part takes the synthetic genetic circuit from Amit et. al. 2011 as well as the NRII promoter and coding region from Amit et. al. 2011 and puts it onto a BioBrick backbone 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. The promoter and coding sequence for the NRII protein (which is mutated to be solely a kinase that can phosphorylate and activate the NRI and allow it to bind to the enhancer region) was taken from the Amit et. al. pACT Tet helper plasmid and put the sequence between the UNS 3 sequence.  NRII was moved to the same plasmid as the synthetic enhancer to 1. reduce the circuit's dependence on LacI (which is also expressed in the original pACT Tet helper plasmid) as well as decrease the interference of these plasmids with the current bacterial circuitry and thus reduce the metabolic strain on the bacteria.
+This part takes the synthetic enhancer circuit with a three TetO binding cassette as well as the promoter and coding region of NRII from the helper plasmid of Amit et. al. 2011 circuits and puts it onto a single BioBrick backbone flanked by the UNS regions. 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. Combining the NRII and synthetic enhancer reduces metabolic strain, decouples the system from LacI/IPTG dependence, and allows for an ease of cloning due to the UNS sequences. The number of filled TetO sites influences the rigidity and the thermodynamics of the looping.
-Once the looping and interaction between the enhancer and promoter happens, transcription of the NRI protein for positive feedback as well as the output fluorescent reporter (mCherry) is initiated.  This part (52s) is from the synthetic enhancer genetic circuits created by Amit et. al where there is a tetR binding cassette with three TetO binding sites in the spacer region between the enhancer and the promoter.  The three binding sites allows for four discrete states of output: a repressed, two intermediate, and unrepressed states. 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 three available TetO binding sites. When all TetO sites are bound, the DNA becomes very rigid 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 two intermediate steps where enough TetR is bound that at any point only one or two TetR are bound to the teto sites and this decreases the flexibility of the DNA and allows for looping and activation and reporter output less frequently.  Finally, when aTc concentration 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.
+This part should be transformed with Bba_K2066022 to get constitutive expression of TetR repressor and allow for the circuit to output a 4 step multimodal response.
-This part should be transformed with Bba_I739001 to get consititutive expression of TetR repressor.
 Source: