1. Lab work safety
1. General considerations
2. Instruments
3. Chemical risk-assessment
2. Biological risks, biosafety rules
1. Microorganisms
1. Host
2. Pseudomonas
2. Biobricks parts used
1. Toggle switch
2. Quorum Sensing
3. RsmA
3. A bacteria went out of the lab, another cell was killed, what happened and how likely would that happen ?
3. 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. 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, mercury should be use in a water solution. It is the form Hg2+ that will be used. To limit the risks we will use a solution already prepared that we will just have to dilute to the wanted concentrations.

the environmental issue of the toxic waste management. Liquid having Mercury or tips and dishes that are in contact with this toxic are kept in specials bins. This rubbish bin is then given to a society specialized into toxic waste treatment. A slip monitoring is sign up by every organism that is involved into the production, transportation and treatment of the toxic waste. When the later is cremated, the producer of the waste receive and attestation that must be kept as a proof of the appropriate treatment. Our project is based on the utilization of mercury, which raises Mercury is an element that has toxic effects on brain and renal function. The other source of important chemical risks is BET. To avoid these latters, experiments are performed under chemical hoods and used contaminated materials are sterilized.

During our project, mercury is conserved in the laboratory and is subjected to special treatment for elimination of heavy metals.

Concerning the reprocessing of toxic chemicals or biological wastes, we follow the standard protocols of our lab. The main toxic wastes we have to deal with in our experiments is BET, the other very dangerous chemical is the mercury. Each one of this product is collected in a special barrel (one for each dangerous chemical) that are recovered by a company specialized in the reprocessing of hazardous wastes. The biological wastes are sterilized in an autoclave by heat and pressure before reprocessing by another company.

We work with a strain of E.Coli designed for lab works : 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 avoid its development if ever it was to make it out of the lab. Those modifications are :

• An inactivated LacZ and rha genes : the bacteria can use neither the lactose or the rhamnose as a source of energy.
• A deletion into a gene coding for an enzyme (pyr E) that produce a matrice required for DNA fabrication (Thymine and Cytosine bases).
• A deletion into the gene that code for an enzyme involved in the fabrication of arabinose, an amino acid component of most protein.

So this bacteria has limited source of energy, needs to be supplied into DNA and protein constituents. Those are such disadvantages that this strain can only grow on highly supplemented medium. We also apply the national lab biohazard policy to each experiment : all the biological waste are collected on a special bin and autoclaved before leaving the lab.

The system we develop needs to be kept off until we want to induce it. In order to achieve that, we extract a post-transcriptional switch mechanism. This system comes from Pseudomonas aeruginosa, a similar one exists in E. coli.

The rsma system of P.aeruginosa controls numerous genes including some virulence factors expressed in some oportunist condition. When activated, it allows the transcription of some genes involved in the creation of a syringe. The later is used to inject some proteins into a targeted cell. E.Coli has not this kind of pathogenic mechanism at all.

The rsma system could somehow interfere with the Csra system of E. coli, which is highly similar and also involved in global regulation mechanism. This has not been tested so far. So what would happen if our strain was to make it out of the lab ? Could our genetic device activate some gene of a wild E.coli strain, or other bacteria ? We have no experimental data to argue for any response. Considering the worst case - the mechanism we use can be effective in wild bacteria - this event would require several extremely low probability events to occur :

• Harmless lab bacteria containing the device should be out of the lab. So far the project is experimental and no bacteria should get out, as describe above.
• The strain we use can not leave on other medium than the one used in a lab (LB agar) so if spread out, it should quickly die.
• Our strain has no competence to build up a pili and share genetic information. So for any of its DNA to be incorporated, it should be from lysed cell, which decrease the probability of transfer.

If incorporated into a wild strain, the risk would be to activate some virulence gene.

• First, it implies that the wild strain has a similar mechanism of global gene regulation.
• Even if there is a similar mechanism, the wild one and the one extracted from pseudomonas would need to be made of similar DNA sequence to interact. A good compatibility of two mechanisms is quite unlikely.
• To become pathogenic, the wild bacteria should also have virulence genes to human, which is not that common. Otherwise its metabolism would be modified without any consequences to human.
• The system we use is originally activated in some specific conditions : when pseudomonas is in contact with a target cell. In which environment would be the lucky bacteria that could have got the sequence ? A rubbish bin, a dump, a river ?... In most of the situations, it would not be an environment on that gives an advantage to the cell that produce virulence factor.
• Given that it is a global mechanism control system, there are always numerous other check point into the cell before modifying its expression. So even if integrated, the rsma system would certainly be either a disadvantage or would get inhibited.

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 : a genus of soil bacteria that fix nitrogen. The cin quorum regulates growth inhibition, expression of some genes that influence nodulation, but no harmful response have been notice so far. It is quite a Rhizobium specific communication system. This bacteria colonises plant cells within root nodules and has never shown any pathogenic sign to human.

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.