1. Lab work safety
1. General considerations
2. Instruments
3. Chemical risk-assessment
2. Biological risks, biosafety rules
1. Microorganisms chassis
2. Biobricks parts used
1. Toggle switch
2. Quorum sensing
3. Pigment
4. RsmA
5. Analyze of a catastrophic scenario
3. Robustness of the device
3. References
4. Optional question

In general considerations, the work in a laboratory requires the use of complex equipment or performing delicate operations. The material, that could be a machine, chemicals or biological material implies the existence of risks. Risks for the goods and for people in the room, but also risks for the environment and people outside the lab. The safety rules and procedures as well as the personal and collective protective equipment are made to minimize the risks by decreasing the probability of an incident to happen.

Half of our team made an internship at the CEA Grenoble. The CEA have a whole department working on safety issues. There is also a special section of people in charge of the security and the safety. It is called FLS: Formation locale de sécurité, we may translate: Local Group of Security and Safety. They ensure the safety of the people who are working in the center and the visitors and also of the goods and the material. The members of our team who made their internship in CEA have assists to safety conferences organised by FLS.

The whole team work now together in another lab, the CIME (centre inter-universitaire de microélectronique), next to the CEA labs and the Phelma school buildings. All team members have met the safety engineer of the labs where we conduct the experiments. He explained us the safety rules to be followed.

At CEA some researchers worked on microsystems devices to detect and quantify these pollutants. They will share their experience and knowledge with us about the way to conduct safe experiments with very toxic chemicals like mercury and also about technical aspects of existing measurement device. We would like to compare our work, our biosystem to “technological only” system that already exist, in terms of precision, sensitivity, reliability, speed and costs.

We plan to present our work and the synthetic biology to a larger public: companies which fund us, school in our villages and town, a conference at “Midi Minatec”, ...

The experiments we made in our project did not require the use of sophisticated equipment. In fact, we only performed very usual operations of microbiology with a commonly used laboratory strains of E.Coli. In terms of experiment the only exotic and hazardous step is the use of mercury. We have used basic devices that we find in every molecular biological laboratory:

Ultra violet lamp:

There is a risk for the eyes and in case of long exposure for the skin, but the UV lamp are only used to take a picture of our gel after electrophoresis, so we are never directly exposed, and it is always for very short period.

Centrifuge:

The main risk is for the material. The centrifuge have to be perfectly balance otherwise the rotor could break. The majority of the centrifuge in our lab have detector that warns the operator in case of bad balance.

Autoclave:

The operation of the autoclave require a specific training

Water bath

The risks might be contamination if bacterial suspension is spillt, a burn risks might exists in some case (high temperature of water)

We aim to design a detector and measurement device for a pollutant in water, like heavy metals. We are actualy working on two versions of this quantification device. One of them involves the use of the MerR sensor for the mercury, and the second one, TetR for the tetracycline. We therefore need to use mercury to test this system. That raises questions about security for the researcher but also for the public and the environment.

We also use for our experiments another toxic chemical, the EtBr (ethidium bromide). However this chemical is commonly used for microbiology that is why we already have rules and installations ready to work with it. We employ the EtBr to make the DNA visible in a gel under U.V exposure after electrophoresis. We do not use EtBr solution while making our gel but we dip the gel in an EtBr bath after the electrophoresis. Due to the hazardous nature of this product, a hood is specially dedicated to its usage. The EtBr and all material that got in contact with it is store in a special trash in the hood.

In order to test our biosensor, we will have to use mercury in a water solution. It is the ionic form Hg2+ that will be used. The mercury is very toxic and mutagenic. To limit the risks in terms of probability of incident and in terms of hazards, we will use a solution already prepared that we will just have to dilute to the wanted concentrations. Moreover a chemical hood will be dedicated to the preparation of such solutions and to the test of our system, just like with the EtBr. A trash will also be dedicated to recover all the wastes in contact with mercury.

Concerning the environmental issue and the toxic waste management, a society specialized in the reprocessing of hazardous wastes recovers the barrels of toxic chemicals. A slip monitoring is sign up by every organism involved into the production, transportation and treatment of the toxic waste. When the wastes are cremated, the lab receive an attestation that must be kept as a proof of the appropriate treatment.

However we performed usual operations of biology and chemistry for which the risks are well known nowadays, our experiments aim to genetically modify a living organisms. And this aspects of the project safety issue is the most important in terms of information sought because it is the less known. Here we are dealing with probability, scenario that may happened and where consequences are mostly uncertain.

In our bio-safety analysis, we try to take into consideration :

• The risk of the chassis bacteria
• The one of each biobrick as well as their combinations in the whole device
• The robustness of the whole device

In the biobricks section we also focus on a specific system that we have extract from Pseudomonas and want to share with the foundation.

We work with a strain of E.Coli designed for lab work : BW25113. This strain is commonly used by students and researchers. .It has no virulence gene, and is therefore a riskless chassis. Furthermore, it has got several genetic modifications that would limit its development if ever it was to make it out of the lab. Those modifications are :

• An inactivated lacZ, ara and rha genes : the bacteria can use neither the lactose, arabinose or the rhamnose as a source of energy.
• A deletion into a gene coding for an enzyme (pyr E) involved fabrication Thymine and Cytosine nucleotides synthesis (which doesn't make the strain auxotroph for theses base though).

These mutations are quite a disadvantage and limit the development of this strain on a minimal media. The main security is still the national lab biohazard policy: after each experiment, biological waste are collected on a special bin and autoclaved before leaving the lab.

The genetic device we develop is a toggle switch which includes two quorum sensing biobricks : cin I and cinR genes. We also use a reporter gene coding for a pigment as an output. At first, we planned to include a post transcriptional regulation mechanism extracted from Pseudomonas aeruginosa. We present here some detail about the toggle and each of those bricks. We found out that the rsma mechanism we develop is the one that needs the most to be analysed. We therefore focused on the later and tried to rationalised the risk that would cause a catastrophic situation. In order to illustrate a series of event, we made an event tree analysis.

Bio-safety is not only about the nature of the basic parts, but also about their combinations. It is necessary to think whether a device might give a dangerous property to any cell. The toggle switch mechanism is a “man-made” specific organisation of inoffensive genetic sequences. It has no hazard on itself but activates the transcription of downstream genes : cinI or cinR. Without any pollutants, only cinR should be transcribed. So they would not be any diffusion of AHL outside of the cell.

An other system used in our device can modify expression of numerous genes : the CinI quorum sensing. The later is shared by many species of legume-nodulating rhizobia (1)⁠, a genus of soil bacteria that fix nitrogen. The cin quorum sensing molecule regulates growth inhibition, expression of nodulation gene, but no harmful response has been noticed so far. It is quite a Rhizobium-specific communication system. These bacteria colonise plant cells within root nodules and have never shown any pathogenicity towards humans and their environment.

We use a combination of three genes that codes for lycopen. This pigment is naturally found in many species of bacterium and has no toxicity.

The Rsma translational regulation system we extracted is from an opportunistic bacteria called Pseudomonas aeruginosa (2)⁠. It is very similar to many others, mostly known as “Csra” that can be found on many eubacteria (3)⁠. The Rsma and csra systems both control a large variety of physiological processes such as central carbon metabolism, motility, biofilm formation, virulence, pathogenesis (4) ⁠and many more (3)⁠... . Basically, when CsrA or RsmA proteins are expressed, they bind to some mRNA leader sequences and act as translational repressors by inducing their degradation. Alternatively, when a small RNA molecule is expressed (called rsmy in the Pseudomonas system) it titres and sequesters the protein, allowing the expression of targeted genes (5). In most systems, the binding site on the RNA leader sequence is a stem-loop containing repeats of GGA nucleotides (4).

In Pseudomonas, the protein RsmA negatively regulates the type VI secretion system, which has been implicated in the P. aeruginosa chronic infections (5)⁠. In some specific environment conditions, rsmY/rsmZ will be transcribed, which produces some proteins that constitute a syringe base plate (4)⁠. The later is then used to inject some proteins into a target cell.

Being originally implied in the activation of some virulence genes, this mechanism implies some safety issues. For instance, the RsmA system could somehow interfere with the CsrA system of E. coli, which is highly similar.

• So what would happen if our strain was to make it out of the lab ?
• Could our genetic device activate some gene in a wild type E.coli strains, or in any other bacteria ?
• Would it, then, become harmful to human or any other organism in the environment ?

As we said above, we want to share the rsma mechanism to the foundation. We therefore though about what could be the most catastrophic situations by using it. The two worse situations we could imagine with an organism containing this brick are:

• an over development of bacteria in any specific environment
• a health threat to human or any other organism

Then we tried to think about what would be the series of event to cause such a disaster. For each of them we tried to think of how probable it is to occur, and made a comment from what we known about.

In order to illustrate our vision of the risk, we made an event tree analysis composed of three colours, each of them symbolise what we think to be a different probability to occur.

On this first table we tried to think about how could the brick containing an rsma system component become harmful to any organism.

Bio-safety is about the safety of the people, the environment, and the system itself. We therefore have to care about how the device would behave if some of the components stop working properly. Any sequence of DNA can get some mutations that would modify its characteristics, or just don't work as planned.

Modelisators of our group tried to test different situations

• if the degradation tag downstream tet R or lacI doesn't work, the toggle switch would still be able to toggle but it would take up to day.
• If the cinI promotor leaks or is not sensitive enough to AHL, the output signal will be hard to distinguish
• If we don't use any post-transcriptional system, the device would still work but be less sensitive.

We could deduce from this last modelling work that the rsma system we develop is not necessary to our device. Given that it is a global regulation system, and that it might interfere with cinI, we decided not to incorporate into our genetic network.

In our project, we can divide the system into four components:

• the toggle switch
• the communication system base on quorum sensing genes
• the output signal
• the translational regulation system based on rsma

Three major questions have been tackled in the following :

• Can the whole circuit or any of its sub-systems cause a safety issue ?
• We use two global regulation mechanisms that can potentially interfere with the chassis metabolism. Could it cause a significant artefact to the measurement of a given molecule ?
• How would be the response to a signal if one of the sub-circuits didn’t' work, or if we don't have time to assemble it ?

The whole system should induce the production of either a Cin I enzyme or an AHL dependent transcription factor. In some condition – or if the toggle doesn't work properly- cells will be able to produce a natural pigment.

Wisniewski-dy, F. and Downie, J.A. Quorum-sensing in Rhizobium. Antonie van Leeuwenhoek 397-407(2002).

Brencic, A. and Lory, S. Determination of the regulon and identification of novel mRNA targets of Pseudomonas aeruginosa RsmA. Molecular Microbiology 72, 612-632(2009).

Timmermans, J. and Melderen, L.V. Post-transcriptional global regulation by CsrA in bacteria. Cellular and Molecular Life Sciences 2897-2908(2010).doi:10.1007/s00018-010-0381-z

Mercante, J. et al. Molecular Geometry of CsrA ( RsmA ) Binding to RNA and Its Implications for Regulated Expression. Journal of Molecular Biology 392, 511-528(2009).

Bernard, C.S. et al. MINIREVIEW Nooks and Crannies in Type VI Secretion Regulation . Society 192, 3850-3860(2010).

Manipulation of living organism allows producing artificial form of life and metabolism. These modifications, although well controlled, require application of the precautionary principle.

Even if in our project we don't plan to take our work out of the lab, so this can not be a cause of safety issue, engineered bacteria might be accidentally or on purpose released in the environment. So, caution involves the implementation of different blocking to limit the propagation of these organisms in the nature:

Nutritional blocking:

Organisms could survive only with artificial substances. In this way, in case of release into the nature such organisms would die.

Evolutionary blocking:

Organisms couldn’t adapt themselves and evolve alone in the nature. This blocking prevents mutations of the organisms that allow them to survive.

Preprogrammed cellular death:

implementation of a suicide gene which is inhibited during wet work. In this way, organisms couldn’t survive outside the laboratory.

In our project, and we think it is the case of many other, the major problem we want to avoid is In case of a bacteria would go outside the lab, efficient methods can be used to limit or prevent their development. Synthetic biology project should employed living organisms mutated to become enable to survive outside the lab. For exmple bacteria that must use amino acids which do not exists in the nature. It is also possible to force the microorganisms to use rare carbon sources. An other possibility is the use of a suicide gene repress by an artificial molecule that can not be found out of a laboratory. Another ways is to make bacteria weak face to the micro-organisms natural selection.

For the researcher’s safety in lab, the work in sterile middle, overall and gloves wearing and all other standard protections things are evidently recommended.

To increase the safety off iGEM competition, we think about bacteria which have an inducible essential gene for binary division by a chemical not existing or rare in nature, by this way the bacteria can’t be divide itself so it will be not selected and going to disappear nearly.