Difference between revisions of "Part:BBa K1400004"

Revision as of 03:10, 18 October 2014

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.

Sequence and Features