Exhibitions: 11360!

Talks: 700!

Workshops : 370!

Conferences: 120!

International Symposium:

2000!

Others: 263!14,000 people

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.

0!

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M1! M2! M11! M12!

Relat

ive Fl

uore

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ce U

nits!

25°C!30°C!37°C!42°C!

Figure 7. Gaussian Function fitting of the experimental data shown in figure 6.

Figure 8. Number of People reached by each of our social activities.

Finally, above 42ºC, the system is shut down (figure 3c), as the cI protein herein used is thermolabile and only functional below 42ºC.Tet and LacI then repress the transcription of mCherry and GFP, respectively. in silico design and chemical synthesis of our parts was performed in submodules as shown in figure 3, which allowed us to test their activity individually using the methdology depicted in figure 4.

RNAthermometers (RNATs) are temperature sensing

sequences that regulate translation by folding into secondary structures that prevent the ribosome 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 fitting our data to a Gaussian function provides us a way to quantify the strength (amplitude), optimal value (horizontal shift), and definition or clearness (width) of our RNAT activity (figure 7). We believe increase in the overall protein degradation rate due to higher temperatures to be the main contributing factor to the negative slope.

While M12 was not temperature sensitive (and is to be sequenced), M1 fulfilled our expectations, with nearly a 10-fold increase in expression at 37ºC relative to 30ºC. For further collective tests, we are to use a RT-PCR thermal-cycler to 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 synthetic biology. This would be especially helpful for the substitution of chemical inducers and alleviation of complex systems. To test this, we set to build a circuit that results in three discrete states whose transition can be regulated by temperature changes only (figure 3). Figure 6. Relative fluorescence of Clones

transformed with module of figure 3b.

UANL-Mty Mexico: ThermocoliAcknow

ledgments W

e would like to thank the School of Biology 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 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

Poor fluorescence was initially observed, but we found that puncturing the microcentrifuge tubes to enhance oxygenation greatly improved our results (we discovered that in fact, mCherry is very sensitive to hypoxia[4]).

Biosafety guidelines according to the

World Health Organization were followed, and the risks our synthetic organisms pose were fully assessed, which included measuring plasmid stability through cultivation until resistance loss (figure 5a) and designing an identification strategy through unique flanking sites (ID-tags, figure 5b), in case of accidental releasement. Our project was approved by the Intersecretarial Commission of Biosafety for OGMs.

Concluding, our results are encouraging as they prove a functional behavior of a crucial module in our genetic circuit. We expect to obtain a similar behavior with the 32ºC RNAT, and finally test if a functional integration of transcriptional and translational regulation can be achieved through this approach.

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

Positive results were obtained from sub-module in figure 3b, to date. Unexpectedly, transformation with the construct yielded clones with different fluorescence intensities (M1,2,11,12, Figure 6), suggesting other factors besides RNAT sequence influence reporter's expression.

Among the many activities classified in figure 8, we created two major didactic exhibitions to massively inform the crowds about synthetic biology, and presented them in public spaces of our city and in rural communities. We also collaborated in the organization of the most innovative symposium of Biological Sciences in Latin America, with a strong focus on synthetic biology, to be held in November 21-23rd, (www.genobiotec13.com).

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).

0%!

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Day 1! Day 2! Day 3! Day 4! Day 5!

Social Impact:

b)

a)

c)BBa_K1140006

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Figure 5. Risk assessment data. a)pUC57 plasmid retention percentage in absence of selective pressure b)ID tags diagram, where unique primer annealing sites are placed before pre/sufix.

b)

a)

37ºC

37ºC