iGEM Paris-Saclay Team: GEMOTE project

Goal

One tool, many applications

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Genetic tools

RNA thermometer

RNA thermometers are sequences located in the 5’ untranslated regions of messenger RNAs with structures that depend on temperature: at low temperature the Shine-Dalgarno sequence (SD) is inaccessible to ribosomes because of the tridimensional folding of the mRNA region containing the SD sequence. At higher temperature, the pairing at the origin of the tridimensional folding is weakened and ribosomes recognize the SD, allowing protein synthesis.

Thermosensitive protein

The cI protein is a repressor from the phage lambda that controls its own transcription and the transcription of the Cro protein. In 1966, a temperature-sensitive (Ts) cI repressor mutant, called cI857, was isolated. At low temperature, this cI857 mutant protein is correctly folded and retains its wild type repressor activity, whereas at nonpermissive temperature (above 42°C), the repressor is denatured and therefore inactive, allowing the transcription of the genes under its control.

Principle of RNA thermometer

Consequences of the folding Narberhaus F, Waldminghaus T, Chowdhury S (January 2006). FEMS Microbiol. Rev. 30 (1): 3–16.

The upshot of all this 4

< 30°C <

Expected plasmid construction

Plasmid construction obtained

After Gibson assembly, transformed bacteria were supposed to contain the following construction and turn red below 32°C and over 42°C.

Sequencing of the construct showed that it only contained the lycopene biosynthetic genes as indicated on the figure above. The fact that the clone turned red indicate that a leaky promoting activity is likely located upstream from the operon, allowing its transcription.

Finally, our colonies followed the pattern:

A test to determine the temperature range in which clones turned red was realized. Colonies were clearly colored after 2 days of incubation.

Clones expressing the lycopene genes were smaller that control E. coli colonies, suggesting some toxicity of the molecule.

Preventing GMOs dissemination

The biggest risk when working with GMOs is accidental dissemination. With our system, if bacteria find their way into the real world by accident, the temperature change will activate the toxin and cause its suicide. So, our system could improve safety in the field of synthetic biology and prevent accidental dissemination of GMOs.

A thermal screening method

Sometimes, we can also wish the death of bacteria. This is usual achieved using antibiotics or auxotrophy. Bacteria carrying this construction could be counter-selected by simply lowering or rising the temperature.

A guardrail for probiotic bacteria

Certain bacteria have demonstrated probiotic effects. It is not hard to imagine that in a near future, probiotic GMO bacteria will be part of our alimentation. Our system could be implemented in such GMO probiotic bacteria. The toxin will not be expressed as long as bacteria are in our digestive track, but it will be expressed when bacteria are evacuated outside of our body due to the temperature outside the fixed range. This will prevent their dissemination in the environment.

A temperature monitoring bacterium

The first application of our project is to assist scientists in their everyday work. The bacteria’s color will indicate if the culture temperature was maintained within a certain range of temperature. Red bacteria will indicate that they were exposed to temperatures outside the fixed range. This range may be modified by introducing mutation in the RNA thermometer sequence.

At this time, the construction that we proposed gives only a visual indication of the temperature at which the bacteria were grown. However, we could imagine others applications; in particular by replacing the lycopene operon by a gene encoding a toxin that kills the bacteria, such as CcdB, Gemote could be like a temperature prison.

Control of the reporter system expression by temperature, thanks to the cI repressor and the RNA thermometer.

By combining this two kind of regulation we propose a new tool allowing a gene expression within a chosen temperature range. Below a given temperature, the repressor is not expressed, while at high temperature, it is inactivated. In order to establish our tool, we use a reporter system in which the lycopene production is under the control of CI. If CI is not active, cell should be red. • Below 32°C, the RNA thermometer prevents CI translation and bacteria will be red. • Between 32°C and 42°C, the cI gene is translated and active, bacteria will be colorless. • Above 42°C, the CI repressor is denatured and therefore inactive, bacteria will be red.

How it works 3

For our first participation in the iGEM competition, we had the idea of creating a new tool that can allow the expression of a gene or an operon in a certain range of temperature. Taking advantage of what already exists, we combined two kinds of temperature dependent regulators : a RNA thermometer and a thermosensitive repressor protein. Their association controls the expression of the gene or operon in the chosen temperature range. The “GEMOTE” (Gene Expression MOdulated by TEmperature) system is born ! Many applications are possible , imagination is just the limit !

Students: Saber BEN MIMOUN, Geoffrey COSTILHES, Thibault DI MÉO, Audrey DOLYMNYJ, William FONTANAUD, Lucille GARNIER, Séverine MAIRE, Jérémie MATHURIN, Doriane MÉNIGAUX, Pierre MOREAU, Yohann PETIOT, Julien RABINOW, Benjamin VIGNARD, Advisors: Solenne ITHURBIDE, Thao Nguyen LE LAM, Instructors: Philippe BOULOC, Sylvie LAUTRU, Jean-Luc PERNODET.

Principle of the thermosensible cI protein.