Difference between revisions of "Part:BBa K1400000"

Latest revision as of 01:53, 19 September 2015

PTRE(4)GX Dual input promoter. Activation at tetO binding sites, repression at gal4 sites.

This is an engineered variant of the pGAL1 promoter native to S. cerevisiae. This dual input promoter has four upstream activating sequences (UAS) and two repressing sequences. The four UAS sites are tetO binding sites that can bind to the tetracycline responsive activator protein, rtTA (reverse tetracycline-controlled transactivator), to induce transcription. The third and fourth GAL4 binding sites of the native pGAL1 promoter are replaced with tetO sites in this version. The Mig1 sequences that are native to the pGAL1 promoter are replaced with two tetO sites. The two repressing sequences are binding sites for the GAL4 DNA binding domain proximal to the TATA box, causing transcriptional repression by steric hindrance and prevention of transcription machinery assembly at the promoter. 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 aTc (anhydrotetracycline). The level of transcription can be modulated or repressed with the addition of β-estradiol.

Figure 1: Characterization of pTREgx via dual drug induction. pTREgx has 4 activating tetO sites and 2 repressing gal4 sites 10bp away from the TATA box. Thus, activation increases with aTc concentration and repression increases with estradiol concentration. Activation is caused by the protein rtTA (reverse-tetracycline transactivator), and repression is caused by the protein GEV (GAL4 binding domain-human estrogen receptor-VP16 activator domain), which is expressed by the strong constitutive promoter, pADH1.

uOttawa 2015

uOttawa created a similar promoter to pTREGx in 2015 that has strong constitutive expression, but still is repressed by GEV. The part is called pPGK1Gx, and is based on the constitutive PGK1 promoter. Find more info on the pPGK1Gx page.

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.

Sequence and Features