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Team:UANL Mty-Mexico/Project
From 2013.igem.org
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.
Although RNATs show almost no sequence similarity among them, a number of structural features can be used to classify them. Here we enlist the most described RNATs structural groups described to date [Kortmann and Narberhaus, (2012)]:
- ROSE.- ROSE stands for "Regulation Of heat Shock Expression". ROSE elements are 60 to >100 nucleotide sequences found upstream of heat shock proteins. They have been found to be conserved in alpha and gamma-proteobacteria. Among the structural features of the ROSE element family are: a) their folding in 2 to 4 stemloop structures; b) a short conserved sequence (UU/CGCU) near the Shine-Dalgarno sequence; and c) the presence of a number of non-cannonical base interactions (the G83-G94 pair; a triple bair among U96-C80-C81; the U79-U97; and the interaction of the AUG codon and C71, G72 and U73. Functional ROSE elements have been found in E. coli (rpoH and ibpA) and B. japonicum (hspA).
- FourU elements.- these elements are characterized by a short motif composed of four uridines that pair with the Shine-Dalgarno region and is embedded in a hairpin that shows temperature-induced conformational changes. FourU elements have only one A-G non-cannonical base interaction. Among the structural features that characterize FourU elements are a) the A-G pair and b) the G34-C46 pair that regulates melting. Functional FourU elements have been described in Salmonella (agsA) and Yersina pseudotuberculosis (lcrF).
- Synechocystis hsp17 element.- with a length of 46 nucleotides, this is the shortest RNAT described so far. The distinctive structural features essential for the function of this element are a) the pairing of a UCCU sequence with the AGGA in the Shine-Dalgarno sequence and b) the presence of two loops in its stems.
- Coding region spanning RNATs.- RNATs are not exclusively found in the 5'UTR of genes; they can also span into the coding region and even be intergenic. Functional coding region spanning RNATs have been found in E. coli (rpoH), phage lambda (cIII) and Lysteria monocytogenes (prfA).
- Cold shock RNATs.- cold shock RNATs also depend on the dynamics of the folding of different loops, but in contrast to heat shock RNATs, the conformation that prevents the binding of the ribosome is found at high temperatures, while at low temperatures, the RNAT folds into a conformation that allows for the ribosome to proceed. An example of a cold shock RNAT is the element found upstream and inside the coding region of E. coli gene cspA.
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.
