Safety proposal

Report of Bacillus pathogenicity

The Bacillus genus is composed of gram-positive bacteria, rod-shaped, aerobic, sporulating ( very resistant spores: heat, dessication etc.). Most of the species are ubiquitous in the environment and only two of them are dangerous pathogens. . Here is a quick overview:

• Bacillus anthracis:

Causative agent of anthrax, rare in industrialized countries (« 1 to 3 cases/yr », ref#2) quite active worldwide (« 20 to 100 thousands human cases per year », ref#2) usually from cattle to humans, ultimate reservoir is the soil, spores of anthracis are very resistant, spores are the contaminants through mostly cutaneous routes (“95% of times” ref#2) sometimes airborne route. Incubation period: 2 to 7 days. Cutaneous form heal itself 80/90% cases however the internal form (rarer) is more dangerous.

• Bacillus cereus:

It is a food poisoning agent. 2 types of syndromes: short incubation and diarrheal (or long incubation). Max 10 day of illness for long incubation syndrome. Self-limiting and benign

• Non-anthracis Bacillus:

Mostly saprophyte (feed of organic non-living matter). Common infections of the eye (all sorts of illness) that can end up in blindness and even loss of the eye. All traumatic wounds, infected burns and any serious lesions can potentially be a terrain for Bacillus strains but it is VERY rare. Bacteremia: very frequent during Bacillus infections but easily eradicated. Generally, produces inflammation, edema and hemorrhage. However, many times when Bacillus strains are found it is in a mixed infection.

• Bacillus subtilis: hypersensitivity reactions observed with contact to autolysates. Symptoms: asthma, skin inflammation (dermatitis), lung inflammation (pneumotitis).
• Treatment: All Bacillus species can be treated with non-ß-lactam antibiotics

References

1. Farrar WE Jr. 1963. Serious Infections Due to “Non-Pathogenic” Organisms of the Genus Bacillus. Am J Med. Vol:34. pp:134-41 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 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. [1]
4. Bacillus on Wikipedia [2]

In the process of review

Bacillus subtilis Final Risk Assessment [3]
Directive 2009/41/EC of the European Parliament and of the Council of 6 May 2009. Contained use of genetically modified micro-organisms. Official Journal of the European Union. pp75-94. [4]
To know what to do for igem wiki igem Safety page

Risk assessment for our specific project

"According to the WHO biosafety is the prevention of unintentional exposure to pathogens and toxins, or their accidental release, whereas biosecurity is the prevention of loss, theft, misuse, diversion or intentional release of pathogens and toxins" (iGEM 2011 Saftey page [5]).
We will not further exacerbate the pathogenicity of Bacillus subtilis, we will not use toxin, potentially dangerous metabolites naturally present in this species. On the contrary, we aim at using external inducers and orthogonal systems thus limiting risks. Furthermore, we will not use cultured cells other that subtilis and Escherichia coli with standard protocols. However, as in any projects involving the use and modifications of living organisms there are inherent problems such as the antibiotic resistance given to certain strains.
Biosecurity is respected since no participants to the Paris 2011 Bettencourt team has any conflict of interests of any kind.

Risk management in the laboratory

There are basic precautions and security procedures that MUST be followed in the laboratory

• 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.