Team:UANL Mty-Mexico/Project

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. Likewise, de novo designed synthetic RNATs have been proved to regulate genetic expression at different temperature ranges. Successful approaches are based on structural design of sequence loops that mask the Shine-Dalgarno (SD) sequence inside the RBS.  Specifically, Neupert et al. (2008) achieved to build simple RNATs that consist of a single small stem-loop structures containing the RBS and blocking the SD sequence. Additionally, 2011 iGEM team BYU_Provo also worked with the design of synthetic RNATs, managing to select sequences sensitive to narrow temperature ranges.

Our project builds upon these two works to prove that RNATs can also be employed to effectively regulate the expression of transcription factors in synthetic circuits. Even when the naturally found RNATs usually regulate the expression of transcription factors, the synthetic constructions made so far have focused mainly on the characterization of the effect of a given RNAT by placing a reporter protein (LacZ or a fluorescent protein) directly downstream. On the other hand, our proposal points at possible applications for the circuit topologies that would be made feasible through the combination of transcription factors and temperature sensitive translational regulation.

Our genetic circuit combines two synthetic RNATs and three transcription factors, through which we intend to gain temperature-mediated control over the transition of three discrete states, signaled by the expression of distinct fluorescent proteins. The figure below shows the topology of the proposed circuit. 

At temperatures below 32ºC, called state 0, no reporter protein is produced. Above 32ºC, the first RNAT is melted thus allowing the translation of GFP transcripts (figure 1a) and the system enters state 1. Note that, despite the fact that GFP transcription is under the regulation of a Lac promoter, LacI is not being produced so far as its translation is prevented by a different RNAT. 

When the system reaches 37ºC, mCherry’s RNAT melts allowing it to be translated (figure 1b) and the system enters state 2. At this point, LacI repressor -whose translation is regulated by the same RNAT- is produced, therefore repressing GFP. Again, note that even if mCherry is transcribed from a Tet promoter, Tet repressor’s transcription is blocked by cI represor. 

Finally, if the system reaches a temperature above 42ºC, the system is shut down (figure 1c). This is achieved because the cI repressor herein used is thermolabile, and above 42ºC it is no longer able to bind its operator and repress transcription. Hence, both LacI and Tet repressors are again produced blocking the transcription of GFP and mCherry, respectively.