Difference between revisions of "Part:BBa K2066038"

Revision as of 12:36, 19 October 2016

Sigma54 Enhancer 57S, no DT, UNS standard

The physical interaction between the assembled sigma 54 promoter complex (glnAp2) and enhancer sites causes the activation of transcription.

Usage and Biology

Amit et. al. generated a genetic circuit regulated by the availability of NRI binding protein as well as the NRII2302 helper protein, which can phosphorylate the NRI protein and activate it as to allow it to bind to the enhancer region. Once this happens, the looping (shown in Figure 1) the DNA from the enhancer to the promoter allows for transcription of more NRI (positive feedback) as well as the fluorescent reporter. When repressor binding sites are placed in the spacer region between the enhancer and promoter, this allows for the regulation of output. The TetR binding makes the DNA looping harder and more rigid, resulting in less transcription of the fluorescent reporter. Multiple repressor binding sites can give rise to discrete number of TetR binding to the region at a given time, thus modulating the ability for the DNA to loop, ultimately giving rise to different states of expression depending on the availability of functional apo-repressor protein.

In this insert, there are two tet binding sites in 57s, allowing for three discrete states of output.

Sequences 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 UNS sequence from Torella et. al.

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 151
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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 ===Usage and Biology===
 Amit et. al. generated a genetic circuit regulated by the availability of NRI binding protein as well as the NRII2302 helper protein, which can phosphorylate the NRI protein and activate it as to allow it to bind to the enhancer region.  Once this happens, the looping (shown in Figure 1) the DNA from the enhancer to the promoter allows for transcription of more NRI (positive feedback) as well as the fluorescent reporter.  When repressor binding sites are placed in the spacer region between the enhancer and promoter, this allows for the regulation of output.  The TetR binding makes the DNA looping harder and more rigid, resulting in less transcription of the fluorescent reporter.  Multiple repressor binding sites can give rise to discrete number of TetR binding to the region at a given time, thus modulating the ability for the DNA to loop, ultimately giving rise to different states of expression depending on the availability of functional apo-repressor protein.
-NRII endogenously has phosphatase and kinase activity, and thus to control the phosphatase activity, the 3.300LG E.coli strain, which consists of a knocked down version of NRII2302, was used to decouple the circuit from a nitrogen dependent PII signal transduction pathway.
+In this insert, there are two tet binding sites in 57s, allowing for three discrete states of output.
-https://static.igem.org/mediawiki/parts/2/2e/T--William_and_Mary--Synthetic_Enhancer_1.png
+Sequences 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 UNS sequence from Torella et. al.
-https://static.igem.org/mediawiki/parts/a/a0/T--William_and_Mary--Synthetic_Enhancer_2.png
-Figure 1.  Adapted from Amit et. al. a. Cartoon representation of how the synthetic enhancer suite works.  The DNA binding NRI-P hexamer has to bind to the enhancer binding sites and this complex can loop (shown to the right) and kinetically bind with the poised promoter to allow for the transcription of NRI and mCherry.  The NRI production allows for positive feedback so as long as it gets phosphorylated by NRII2302 (expressed by pACT Tet helper plasmid), it can bind to the enhancer sequences and continue expression.  Adding TetR repressor binding sites between the promoter and enhancer regions can weaker the probability of the looping event and reduce the expression of NRI and mCherry at a given bound state.
-Unlike the traditional model of repression, this circuit does not use the repressor to block or compete for the RNA polymerase binding site, but rather decreases the likelihood for the enhancer to loop and bind to the promoter region.
-To affect the looping probability, Amit et. al. cloned in TetR repressor binding sites, TetO sites, within the Spacer region (region between the NRI binding sites and enhancer which promotes the looping and activation of transcription).  The binding of TetR causes DNA to become more rigid as to weaken the looping process and decrease the quantity of output depending on how many repressor proteins are bound to the TetO sites at a given time.
-Amit et. al. used small molecule induction with anhydrous-tetracycline (aTc) to modulate the conversion of a variable induced concentration input into discrete or step like output expressions via discrete number of TetO binding sites between the promoter and enhancer, affecting looping propensity. For example, in Figure 2, Amit et. al. generated a synthetic enhancer suite that has three TetO sites between the promoter and enhancer. Conceptually, these three sites, should allow for four different states of expression: 1. Completely repressed state when there is almost no aTc in the cell to block the TetR from binding and repressing the looping, 2. An intermediate step where there is enough aTc to bind to TetR as to allow for an average of 2 TetRs to be available to bind to the TetO sites and partially repress the expression, 3. A second intermediate step where there is more aTc in the cell than the former situation as to allow for only very few TetR being active and available to bind and repress the circuit and finally 4. Enough aTc to reach the saturated, unrepressed state of the circuit where there most of the aTc is bound to a TetR, making it functionally inactive to bind to TetO and repress the circuit.
-https://static.igem.org/mediawiki/2016/1/1e/T--William_and_Mary--Synthetic_Enhancer_Induction_Curve.png
-Figure 2. Graph adapted from Amit et. al., showing the three discrete steps of the transfer function once three TetO binding sites were placed between the promoter and enhancer, affecting the looping and transcriptional activation propensity. There are 4 discrete steps, consecutively shown on the graph as the aTc concentration increases logarithmically as a completely repressed state, two intermediate states, and an unrepressed state.  The data was plotted on the y-axis as the ratio of the fluorescence level measured in the presence of a given aTc concentration divided by the maximum fluorescence level.
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