Part:BBa_K1400004
pGALtx Dual input promoter. Activation at gal4 binding sites, repression at tetO sites.
This is a dual input promoter that can be activated and repressed by two different activating proteins (Tet and Gal4). Is based off the Gal promoter in Saccharomyces cerevisiae, which is a relatively strong promoter. It has four gal4 sites upstream of the TATA box, which can be bound by Gal4 binding proteins or phusion activators like GEV (Gal4 binding domain, human estrogen receptor, vp16). 10bp downstream of the TATA box two tetr binding sites are added. These can be bound by the tet repressor, or by activating variants using the Tetr binding domain like rtTA (tet binding domain, vp16 activating domain).The close proximity of these sites to the TATA box cause any binding protein (activating or repressing) to repress expression. The close proximity of these sites all seem to affect transcription rates of this promoter, as it has significantly less expression than the native Gal promoter. Thus this promoter can be used for complex control of expression
In cells expressing rtTA and GEV (GAL4 binding domain-human estrogen receptor-VP16 activator domain), this promoter can be used to drive transcription of a downstream gene by the addition of β-estradiol. The level of transcription can be modulated or repressed with the addition of aTc (anhydrotetracycline).
Figure 1: Four activator site pGALtx under different repressor saturation. pGALtx has 4 activating GAL4 sites and 2 repressing tetO sites. On the left, rtTA is driven by the weak constitutive promoter pMRP7 and on the right rtTA is driven by the strong constitutive promoter, pADH1. aTc represents the amount of functional repressing activator, while estradiol represents the amount of functional activating activator.
When we first characterised these promoters, we were using a weaker constitutive promoter to drive the repressing activator. By using a strong repressor we got dramatically increased repression, indicating a certain saturation point is required of activator for repression to be robust.
This promoter was originally designed and tested by Tom Ellis et al. 2009.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 367
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 76
- 1000COMPATIBLE WITH RFC[1000]
uOttawa 2015
The original use case for this part was to use it in a tri-stable switch (see uOttawa's [http://2014.igem.org/Team:uOttawa 2014] and [http://2015.igem.org/Team:uOttawa 2015] projects for more details about this network). However, further analysis of our mathematical models showed that the dynamics exhibited by this promoter cannot lead to tri-stability.
Specifically, this promoter behaves multiplicatively. This means that adding a fixed amount of repressor will down-regulate the gene based on how much it is induced. For instance, adding a certain amount of repressor will cut the promoter's expression in half, rather than lowering it by a fixed amount. This relationship can be modelled roughly by the following expression:
d/dt [GFP] = a + f([GEV]) * g([rtTA]) - b[GFP]
where 'a' is the basal expression rate, 'b' is degradation rate, and 'f', 'g' are some binding functions. We used Hill dynamics in our models. Notice how the binding for GEV and rtTA are multiplied together. What is necessary for tristability is 'additive' dynamics:
d/dt [GFP] = a + f([GEV]) + g([rtTA]) - b[GFP]
See our [http://2015.igem.org/Team:uOttawa 2015] project for more details.
The promoter also seems to behave differently when doxycycline is used instead of aTc to modulate repression by rtTA. With doxycycline, repression seems to be much weaker and activation with beta-estradiol modulating GEV seems to have a greater effect on the activity of the promoter.
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