0!

0.1!

0.2!

0.3!

0.4!

0.5!

0.6!

M1! M2! M11! M12!

Relat

ive Fl

uore

scen

ce U

nits!

25°C!30°C!37°C!42°C!

Figure 6. Relative fluorescence of Clones transformed with submodule of figure 3b.

Positive results were obtained from submodule 2 (figure 3b), to date. Unexpectedly, transformation with the construction yielded clones with different fluorescence intensities

Exhibitions: 11360!

Talks: 700!

Workshops : 370!

Conferences: 120!

International Symposium:

2000!

Others: 263!

a) b)

Figure 1. Predicted secondary structure of the a) 37 ºC and b)32 ºC responsive synthetic RNATs.

Figure 2. Temperature dependence of mCherry’s translation by the 37ºC RNAT in E. coli. a)Control 30ºC b)Control 37ºC c)M1 clone 30ºC and d)37ºC.

Figure 3. Diagram of our genetic circuit. a)submodule 1 b)submodule 2 c)submodule 3.

Finally, above 42ºC, the system is shut down (figure 3c), as the cI protein used herein is thermolabile and only functional below 42ºC.Tet and LacI then repress the transcription of mCherry and GFP, respectively. Submodules 1 and 2 (figure 3a,b) were tested using the methdology depicted in figure 4.

RNAthermometers (RNATs) are temperature sensing sequences

that regulate translation by folding into secondary structures, which prevent the ribosomes from binding the transcript until higher temperatures shift it to an open structure. We implemented two synthetic RNATs that allow translation above 37ºC [1] and 32ºC [2] (figures 1, 2)

We found that the relation between the saturation level of fluorescence and the temperature can be reliably fitted to a Gaussian function, which illustrates the strength (amplitude), optimal value (horizontal shift), and clearness (width) of our RNAT activity (figure 7). It is also predictive for non-verified temperatures and even distinct RNATs.

smaller plasmid concentrations than M1 and M2 (figure 8). Also, M12 is statistically different from M1 and M2 (p<0.05). Encouraged by these results, we are to use qPCR to both verify PCN and preciselly tune temperature and measure fluorescence at once [3].

To integrate transcriptional and translational regulation is our goal, as it widens the spectrum of potential genetic circuit topologies for information processing in synthetic biology. This would help to substitute the use chemical inducers and alleviate complex systems. To prove this, we set to build a circuit that results in three discrete states whose transition can be controlled by temperature changes only (figure 3).

UANL-Mty Mexico: ThermocoliAcknow

ledgments W

e would like to thank the College of Biological Sciences at UAN

L, our instructors and advisors, and the Herm

anas Rocha y Sida Foundation for the advancem

ent of Science for supporting this project.

[1] Neupert J et al. (2008) Nucleic Acids Res, 36:e124 [2] TUDelft iGEM team (2008) http://2008.igem.org/Team:TUDelft [3] Utermark J and Karlovsky P (2006) BioTechniques 41:150-154 [4] Regmi KC et al. (2013) J Phys Chem B 45:9639-9647

identification strategy based on unique flanking sites (figure 5a) and measuring plasmid stability through cultivation until resistance loss (figure 5b).

Concluding, our results are encouraging as they prove a functional behavior

of a crucial module in our genetic circuit. We found that PCN is the potential cause of phenotypic variation among clones, which could be exploited for regulatory purposes along with RNATs. Further tests will be carried out in order to verify this hypothesis, reproduce the 32ºC RNAT behavior in our construction, and finally test if the full system results in a functional integration of transcriptional and translational regulation.

we reached ∼14,000 people through our human practices. Among many activities, we created two major didactic exhibitions to massively inform the crowds about synbio, (figure 10). We also organized the most innovative symposium of Biological Sciences in Latin America (www.genobiotec13.com). Finally, we fomented the organization of team iGEM CIDEB HSD, and became their instructors and collaborators. We are proud to say they came 2nd runner up, twice in a row.

Below 32ºC, no reporter protein is produced (state 0). Above 32ºC, the first RNAT is melted thus allowing the translation of GFP transcripts (figure 3a), entering state 1. At 37ºC, mCherry’s and LacI’s RNATs allow them to be translated (figure 3b) and GFP is repressed (state 2).

Figure 4. Methodology used to measure fluorescence expression responsiveness to temperature.

Figure 10. Number of People reached by each of our social activities.

Figure 8. Plasmid DNA concentration by clones (ng/uL). 22 assays were carried out, including ∼7 repetitions per clone.

Figure 9. Gaussian Function fitting of the experimental data shown in figure 6.

We suspected variations in plasmid copy number (PCN) to be the cause of phenotypic discrepancies among clones. To test this hypothesis, we developed a simple methodology to estimate PCN (figure 7). M12 clone appears to have consistently

Figure 7. Methodology developed to indirectly estimate PCN.

We simulated the evolution over time of the fluorescence emission at different degradation rates (δ). Results are plotted in 3-axis: time (min), temperature (°C) and fluorescence (RFUs) (figure 10).

Figure 5. Risk assesment data. a) ID tags diagram, where unique primer annealing sites are placed before pre/sufix. b) Plasmid retention percentage in absence of selective pressure.

Figure 10. Simulations for clones M1 and M2 at different values. a) δ=0.1 b) δ=0.01 c) δ=0.001

b)

a)

c)

b)

a)

c)

BBa_K1140006

BBa_K1140004

BBa_K1140002

BBa_K1140003

37ºC

37ºC

pTet

a)

b)

The risks our synthetic organisms pose were

fully assessed, which included designing an

(M1,2,11,12, figure 6), suggesting other factors besides RNAT sequence influence reporter's expression.