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Safety

We are following the official iGEM safety instructions as well as the updated questions sent to us.
We are working in the [http://www.necker.fr/tamara/pages/people.html U1001 research unit] which is shared between INSERM (National Institute of Health and Medical Research) and [http://app.parisdescartes.fr/cgi-bin/WebObjects/Labs.woa/wa/showInfoLabo?cle=20101353#ficheLabo Paris Descartes University]. It is fully equipped for bacterial genetics and molecular biology work in terms of material and people having the competence to do the research. The lab is monitored on regular basis by security officers of both the Paris Descartes University and the INSERM.
Our team is under daily supervision of primarily Ariel Lindner, Senior Research Scientist in U1001. We receive regular help as well from Chantal Lotton (Engineer and Hygiene and Security Correspondent) as well from two Ph.D students Yifan Yang and Antoine Decrulle, both working on site. The whole lab team is also here at times to explain and advise us.

Would the materials used in your project and/or your final product pose:

a. Risks to the safety and health of team members or others in the lab?

Bacillus subtilis is a gram-positive bacteria, rod-shaped, aerobic, sporulating ( very resistant spores: heat, dessication etc.). It is ubiquitous in the environment and it is not usually a human pathogen. It is considered a Level 1 Biosafety Containment Agent which goes according to the following definition: "Agents of no or minimal hazard under ordinary conditions of handling" [http://www.unm.edu/~sheaweb/sheamanual/biosfty/BIOSAFA-pg3.01-A7only.htm].
Hypersensitivity reactions have been observed in the litterature with contact to B.subtilis autolysates. Symptoms: asthma, skin inflammation (dermatitis), lung inflammation (pneumotitis) but it is very rare hence manipulation with gloves and a lab coat is respected.

Escherichia coli: we use DH5a and K12 which are common laboratory strains[http://www.openwetware.org/wiki/E._coli_genotypes], also considered as Level 1 Biosafety Containment agent.
In our different sub-projects, we will not further exacerbate the already weak pathogenicity of Bacillus subtilis: our different designs are aimed at proving the existence and caracterising the nanotubes. It ensues from fundamental research that was published earlier this year. It focuses on a natural phenonemon that B.subtilis seems to exhibit in period of stress.
To this end, we are going to use different antibiotic resistance as well as Green Fluroescent Protein variants to observe under the microscope the transfer of molecules. These two first designs use non conjugative plasmids. Albeit their resistance they do not pose any risks to the safety and health of the team/lab members since the constructs are under IPTG induction.
Other, mainly amplifiers in emitter-receptor systems or fluorescent proteins to monitor the expression of genes (See the Designs page) are used in the other sub-projects of characterization. Hence, we will not use or modify toxins (that B.subtilis does not naturally have [http://epa.gov/biotech_rule/pubs/fra/fra009.htm]) nor any potentially dangerous metabolites that could be present in this species. On the contrary, we aim at using external inducers such as hyperspank promoters (IPTG inducible) and orthogonal systems (such as T7 promoters) or even non natural systems (T7 amber suppressor tRNA) thus limiting risks to both the team and lab members .
In the very unlikely event (see section above) of an infection by either bacterium, a treatment with regular antibiotics is sufficient, antibiotics that are also natural.

References for risk assessment

1. Farrar WE Jr. 1963. Serious Infections Due to “Non-Pathogenic” Organisms of the Genus Bacillus. Am J Med. Vol:34. pp:134-41 [http://www.sciencedirect.com/science?_ob=MiamiImageURL&_imagekey=B6TDC-4BXGKV2-1B4-1&_cdi=5195&_user=658968&_pii=0002934363900470&_check=y&_origin=&_coverDate=01%2F31%2F1963&view=c&wchp=dGLzVlz-zSkzS&md5=5378af9f1b74b03445c94b3a24f99ac6&ie=/sdarticle.pdf PDF ]
2. Farrar WE , Reboli AC. 2006. The Genus Bacillus-Medical. In: Dworkin M, Falkow S, Rosenberg E, Schleifer KH, Stackebrandt E, editors. Prokaryotes, Part 1, Section 1.2. New-York: Springer. 609-630 [http://www.springerlink.com/content/mp23l6460306188q/fulltext.pdf PDF]
3. Turnbull, PCB. 1996. Bacillus. In: Baron S, editor. Medical Microbiology. 4th edition Chapter 15. Galveston (TX): University of Texas Medical Branch at Galveston. [http://www.ncbi.nlm.nih.gov/books/NBK7699/]
4. Bacillus on Wikipedia [http://en.wikipedia.org/wiki/Bacillus]
5. Bacillus subtilis Final Risk Assessment [http://epa.gov/biotech_rule/pubs/fra/fra009.htm]
6. [http://www.openwetware.org/wiki/E._coli_genotypes E. coli genotypes]

b. Risks to the safety and health of the general public if released by design or accident?

None of our designs have potential to harm the public if released by design or accident and even less since our lab is especially equipped for microbial manipulation and everything is done to avoid risk of release.
However, we are conscious that as any bacterium it can be hazardous. For instance, all traumatic wounds, infected burns and any serious lesions can potentially be a terrain for Bacillus strains but it is very rare. When found, B.subtilis is usually mixed with other types of bacteria, it can cause a bacteremia but it is easily eradicated.
Treatment: subtilis like every soil Bacillus species can be treated with non-ß-lactam antibiotics and Escherichia coli can be treated with regular antibiotics, which does not pose any problem to a doctor.

c. Risks to environmental quality if released by design or accident?

None of our design or parts are hazardous to the environment since they mostly code for un-natural systems or detection molecules. If anything they would have a disadvantage for overproducing useless metabolites such as GFP, their competitiveness is decreased. Any kind of horizontal transfer risks is taken into acount with the use of non-conjugative plasmids and under external inducers.

d. Risks to security through malicious misuse by individuals, groups or states?

Biosecurity is respected since no participants to the Paris 2011 Bettencourt team has any conflict of interests of any kind. In addition, there could not be security concerns due to a malicious use of our designs and parts/devices since they only aim at visualising a natural phenomenon present in B.subtilis, it remains fundamental research with no potential misuse outside the laboratory.

Final Statement on Biosafety and Biosecurity

In conclusion, not a single part nor a single system/final product is known to be infective/toxic/pathogenic to humans or threatening to the environment; there would be hence no implications in case of release.

Under what biosafety provisions will / do you operate?

a. Does your institution have its own biosafety rules and if so what are they? Provide a link to them online if possible.

Our institution, INSERM, provides to its researchers, engineers and technicians a set of of regulations, advice and mandatory practices for each aspects of the laboratory life. Here is the link to all the online documentation:
http://www.rh.inserm.fr/INSERM/IntraRh/RHPublication.nsf/mDisplayMotsClefsWeb?OpenForm&arg1=6&arg2#
You can find the relevant information in the tab "Formation - Documentation". Our apologies for the site being in French but you can have some of the safety and advice sheets in English by clicking [http://www.rh.inserm.fr/INSERM/IntraRH/RHPublication.nsf/vMotsClefsWebByCat/0735F4BC31ADA4BEC125756A005246A2/$file/Anglais.zip?OpenElement on this]. In essence, there are information and rules concerning cleanliness and important reflexes to have; it treats on how to sterilize and wash contaminated material. There is also a part concerning hazardous situations (electrical, chemical, ionizing, fire etc.) and how to deal with them, who to contact, how to deal with dangerous situations as well as your rights and obligations such as the one to refuse to enter your workplace if there is potential threat to your place and the obligation to contact the director and the Head Safety Officer of the INSERM that will emit the final judging. A good point is always to tell your hierarchy superior and the lab security officer.

b. Does your institution have an Institutional Biosafety Committee or equivalent group? If yes, have you discussed your project with them? Describe any concerns or changes that were made based on this review.

The iGEM team as a project, is part of the four-year work plan of our hosting U1001 INSERM lab that was reviewed for its scientific merit and safety of the research plan. There was no specific contact with INSERM/University institutional biosafety personnel regarding our specific project that was reviewed by the lab heads and accepted by the lab safety officer - it is within his responsibilities as the representative of the institution to provide such agreement after reviewing our project.

c. Will / did you receive any biosafety and/or lab training before beginning your project? If so, describe this training.

We received a laboratory training prior to the start of the experiments.
The information about biosafety in the lab presented here have been gathered during the introduction made by the Correspondant of Hygiene and Security ([http://www.necker.fr/irnem/units/search.php?iduser=1950&pass=1 Chantal Lotton]) to the safe use of each instrument and machine as well as general guidelines of good lab practice. All the students in the team participated to this introduction. The handling of waste, how to keep things sterile, how to use the hood etc. was also explained to us.
In our team, there are also two designated lab managers that are notably in charge of knowing the functioning of each apparatus and of the security issues; team's students had, as a mandatory part of their undergraduate curriculum, a Hygiene and Security course. In case any problems arise Chantal Lotton, the lab's security officer, can be contacted 24H/24H to seek help and/or advice. Furthermore, weekly team meetings with our supervisors allow for any concerns to be raised and treated.

d. Does your country have national biosafety regulations or guidelines? If so, provide a link to them online if possible.

France, as a member of the European Union follows the Directives of the European Parliament (such as this one [http://www.bmwf.gv.at/fileadmin/user_upload/forschung/gentechnik/2009-41-EC.pdf Directive 2009/41/EC on the contained use of genetically modified micro-organisms]). We, and the research unit we work in, apply the general guidelines that are outlined notably in the aforementioned Directive notably in terms of waste management and sterility.

Safety at work

There are basic precautions and security procedures that MUST be followed in any laboratory and here are some that we gathered from this introduction and from later meetings with the security officer.

• The ten general rules :

Rule n°1: If you're not sure of anything: ASK !
Rule n°2 : Don't smoke or eat or drink in the lab.
Rule n°3 : Don't wear scarves and tie your hair back if needed. Wear coat when it’s not so hot.
Rule n°4 : Don't leave any personal belongs (phone...) because of thiefs.
Rule n°5 : Don't work ALONE at night and during the weekend (and inform the on-call guard of your presence).
Rule n°6 : Be careful, products may be harmful even if they seem inoffensive.
Rule n°7 : Identify your stuff (iGEM + date + if sterile) and CLEAN UP once you've finished.
Rule n°8 : Tell us if something is running out so that we can buy them in advance.
Rule n°9 : Close both lab doors before leaving.
Final rule : Turn off all the machines before leaving (especially the bain marie, 
because of fire and surtension problems in this lab!). Check that -20°C and -80°C are OK every morning.

If you've received chemical spatter on your eyes or your skin, use ONLY water to clean them (at least 10 min under the water)! No detergent!

• Trash can :

Solid carton: Press down and compress!
Black: For everyday use – paper, plastic, enveloppe...
Cardboard yellow box: Biological stuff (Petri dish closed, pipette on verticle angle)
To close the box: Read the instructions on the box, identify it (U1001 + date), then put it in the hallway
Plastic yellow box: Sharp/ cutting items (needles, glass...)

Liquid : Methanol and other: chemical waste bottle

• BET bench :

ASK Chantal before using - Cancer Risk (UV and BET)!
Wear nitrile blue or purple glove, long-sleeved blouse and goggles
No BET in electrophoresis, only in tray.
Use only dishes, pipettes and trash can for BET.
Buffer : 0,5X (be careful if 1X = crystal) TBE (or TAE)

Specials trashes for EtB: Solid (gel, gloves, contamined stuff) in big white box (under the bench)
Tips in yellow box on the bench “EtB”
Liquid: in 5L plastic bottle on the bench (decontamination with 2 bags of charcoal)

• Lavery :

In Dirty stuff Box (Buffer …): Rinse bottle and take out scotch
In Contaminated stuff Box (bacteria …): Let it in the box. (It will be wash with Javel water)
If you decontamined with Javel water, wear blouse.

• Microscopy :

ASK MING for a briefing before using! (his holidays: 2-17 of July)
Verify schedule before entry in the room. Do not let the door open (night for experiments which are on, air conditioned room)

• Machines instructions :
  

Autoclave :
To be used only by COMPETENT AND AUTHORIZED PEOPLE : Kevin and Daniel < risk of burning and glass breaking.

Always UNSCREW the bottles before introducing them into the machine!
Do not try to open during each cycle. Check temperature (85°C) and pressure of autoclave (1 bar) before opening.
Be careful, the top is very hot (high risk of burning yourself, 85°C when opened): Use mittens to take out the sterilized bottles! Check if the scotch lines turned black.
If it's LBA bottle, mix it after sterilizing.

Micro-waves :
Always UNSCREW the bottles before introducing them into the machine!
BIG RISK OF BURNING: Use mittens to take out the bottles!

Washing machine :
To be used only by competant and authorized people (Kevin and Daniel) < risk of burning and glass breaking.
Check the level of water before use (the reserve as to be full). Do not try to open during each cycle.

Osmosed water recipient :
Be careful to close it well (90°) to avoid flood.

Precision balance :
Prepare what you need and close doors everytime before weighing. Clean after use.

Shaker-Incubator :
Rack has to be stable (check that black screws are on tight)
Don't hesitate to launch a new one if necessary.
Sticky band : don't put your hand on it (there is polystyrene stuff on it). To wash it, use only water + soap.
Always incline your falcon and unscrew it a little (to let the air go inside)

37°C and 30°C Incubators :
Be careful when closing the door, it is fragile !
Petri dishes with gelose has to be put on the top (to avoid deshydration)

Hood :
It only protects the area under the hood and not us ! NO PATHOGEN INSIDE!
Clean your hands. Clean the work area with 70% ethanol BEFORE and AFTER use.
(ethanol in 5L bottle (kitchen) + osmosed water)
Try to occupate only middle of area in order to share with someone else.

Centrifuges :
Don't forget to put the hat and to BALANCE out your tubes (weight and symmetric position).
Once you've finished, shut the machine down. Leave the top open.

Freezers :
Use the -80°C only for long term stocks. Use -20°C for working stocks.
Don't leave the door open when you looking for a strain in the boxes.
Close the door of the “kitchen”: this room is air conditioned because of the -80°C!
Use cardboard boxes for storage (not plastic racks).
Always check the temperature and if there is a problem of cut, do NOT open the door (years of research are inside...)

If there is any problem: 2255 (technical service of the faculty: you will join Jean Marie / Florent electricians or Eric (boss)).

4°C - Powder of IPTG, Antibiotics … : Use glove

Burner :
Sterilizes an area of 30 cm radius around the burner. Don't speak when working within the sterile zone, otherwise you'll spread your bacteria !
Be careful at your hear

Bain Marie :
DO NOT FORGET to turn it off once you're done (high risk of combustion!)

PCR / Thermocycler :
Don't let it work at 4°C overnight as much as possible (electricity surtension)! Shut it down after use.

Biophotometer :
1/10 dilution in 50 microL. Do not forget to shut down and close the chamber with the black cap.

Spectrophotometer and Tecan :
Turn them off before leaving.

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