Team:UANL Mty-Mexico/Project

RNA thermometers (RNATs) are RNA sequences that range from 40 to more than a 100 nucleotides commonly found in the 5' untranslated region of some genes and that regulate in cis their translation without the need of other factors [Kortmann and Narberhaus, (2012); Narberhaus, (2009)]. These RNAT sequences show certain three dimensional structures, some of which interact with the ribosome binding site (RBS) of their regulated genes and hinders the proccessivity of the ribosome complex at certain temperatures. The dynamics of the formation of these structures is temperature dependent and is the basis of the regulation of the translation rate of a given transcript [Chowdhury, S., et al.,(2006); Narberhaus, F., et al.,(2006)].

Functional RNAT have been found in different organisms, mainly pathogenic bacteria, and many others have been predicted in almost everyfrom a number of bioinformatic studies. They have been found to regulate the expression of virulence factors, heat and cold shock response factors and even proteins involved the development of some bacteriophages.

Their apparent widespread presence in living organisms has made RNATs attractive for some applications, specially the ones related to the replacement of chemical inducers and for the development of new drugs.

However, from the experience of those who have been working extensively with RNAT in the later years, the accurate bioinformatic prediction of functional RNAT has proven to be an exceptionally difficult task; the reasons for this are pointed to be the poor sequence conservation observed among RNATs and the gaps in our current understanding of the RNAT function, their structural diversity and the effect of other regulatory sequences far from the RBS region [Kortmann and Narberhaus, (2012); Waldminghaus, et al., (2007)].

The discovery of new RNATs has relied on a mixed approach that involves bioinformatics and experimental validation, as well as approaches that involve mutational libraries, synthetic constructions and directed evolution.

Even when the naturally found RNATs usually regulate the expression of transcription factors, the synthetic constructions made so far have focused mainly to characterize the effect of a given RNAT using a reporter protein (LacZ or a fluorescent protein) directly downstream of a RNAT. In our work, we intend to prove that RNATs can also be employed to effectively regulate the expression of transcription factors in synthetic circuits and point at possible applications for the circuit topologies that would be made feasible with this new kind of synthetic regulatory device.

The present project is about a system that involves the use of two synthetic RNA thermometer and expresses different fluorescent colors indicating 3 states of temperature. This system is controlled by temperature and it expresses fluorescence to indicate certain temperature by ranges. We designed different parts which work in a genetic circuit as it is explained in the following:

Circuit description

The circuit has 3 main states, the “Normal” state, the “Green” state from 32ᵒC to 37ᵒC, and the “Red” state from 37ᵒC to 42ᵒC. The system works in the following way: At temperatures below the 32ᵒC the system is in the “Normal” state. After the 32ᵒC the riboswitch changes its conformation and translation can occur. Under these conditions the GFP is expressed indicating that the temperature is higher than the 32ᵒC. The first part [http://parts.igem.org/Part:BBa_K1140002] works with a pLac promoter. This promoter can be repressed by the Lac protein. The GFP has a LVA degradation tag in order to degrade the green fluorescence when changing the “state” of the circuit after being repressed by the LacI.

In the next state two parts are involved: The first one [http://parts.igem.org/Part:BBa_K1140006] works with a repressible promoter pTet and with a 37ᵒC riboswitch producing the RFP with a LVA degradation tag.

The next part [http://parts.igem.org/Part:BBa_K1140003] works with a constitutive promoter and a 37ᵒC riboswitch producing the Lac protein that represses the pLac promoter stopping the GFP production in order to only show the red fluorescence.

Finally, we have two other parts: One part is a cI repressor protein with a constitutive promoter and then we have a LambdacI promoter producing the TetR repressor [http://parts.igem.org/Part:BBa_K1140004]

The cI represses the pLambdacI but its production stops at 42ᵒC, this means that at this temperature the TetR repressor is produced and it inhibits the pTet promoter repressing the RFP production.

 

In summary, from 0ᵒC to 32ᵒC the system shows no fluorescence. The GFP production starts at 32ᵒC up to the 37ᵒC, when the Lac is produced and represses the pLac and the GFP. Also, at this temperature the RFP is produced. At 42ᵒC the cI production stops, the TetR is produced repressing the pTet and no fluorescence is shown then.