New Promoters Library

Description

Transcription is the first step of genetic expression and thus is one of greater and important processes of life. Promoters are DNA sequences that controls the transcription initiation and mRNA production of all genes, recognized and synthetized, respectively, by the enzyme RNA polymerase.

Different regions for interaction of the promoter with RNA polymerase were described as represented in the figure 1 bellow, such as: up element, sequences of -35 and -10 hexamer, extended -10 element, discriminator.

Figure 1 – Specific sequences for interaction between promoters sequences and RNA polymerase

Together, these regions specify the RNA polymerase recognition and interaction to the promoter. Furthermore, two others elements control the transcription of certain gene or set of genes: the repressor proteins and its operators. Many steps of this process can be also highly regulated by accessory protein factors and small ligands, temperature, salt and solute concentrations, and other environmental variables.

About Operon Mer, it’s regulated by MerR repressor protein that, in the absence of ion mercury, binds on a promoter-operator-MerR complex, in which the RNA polymerase is prevented from recognizing the promoter sequence, blocking genetical transcription. However, in the presence of ion mercury, it dislocates from the promoter region and binds to it, being allosterically altered, as represented below.

Figure 2 – Mer Operon regulation performed by MerR repressor protein.

This unique system of genetic regulated expression sensible to the mercury absence and presence, inactivating and activating, inspired us to design new promoter sequences, merging the well documented and widely used promoter sequences with mer operator. We designed 8 new promoters, the first and only collection of MerR regulated promoters, sensible to mercury, of iGEM!

The new designed promoters were merged with Tac promoter and JK26 promoter, promoters that interact with RNA polymerase on exponential and stationary phase of bacterial growth. Check out the synthetic genetic circuits on the videos below.

We characterized its efficiency through the RFP regulated expression! Check this out in results!

Results

To characterize the intensity of the promoters library expression, RFP (BBa_K081014) was linked to them, as reporter gene. Fluorescence tests were performed in parallel to growth curves. From these promoters, the ones who stood out were BBa_K2123006 and BBa_K2123007, but the most well regulated one, chosen to compose Mer Operon, was BBa_K2123004.

Despite not composing Mer Operon, the regulated promoter whose regulation stood out was BBa_K2123001, with overlap operator. At fluorescence tests, comparing to the control, 99% of RFP expression was inhibited, thanks to the design which overlapped not only to spacing, but the isomers -10 and -35, complicating, or almost obstructing, the interaction between RNA polymerase and promoter.

Another promoter which exceled during inhibition tests was BBa_K2123002, in which design between two main isomers, showed 92% inhibtion of RFP expression when compared to control.