Contents 1 Safety Considerations 1.1 University Regulations 1.1.1 PEALS 1.2 Training 1.3 Bacterial Strains 1.3.1 E. coli (DH5a & XL-1Blue) 1.3.2 B. subtilis (168 & BSB1) 1.3.3 B. subtilis L-form 1.4 Bio-Hazards 1.4.1 Ultraviolet (UV) Radiation 1.4.2 Isopropyl β-D-1-thiogalactopyranoside (IPTG) 1.4.3 Chloramphenicol 1.4.4 Ethanol 1.4.5 MiniPrep Reagents 1.5 References

Safety Considerations

University Regulations

Our university has an Occupational Health and Safety Service available which aims to maximise biological safety throughout research and teaching laboratories within Newcastle University. If any enquiries or concerns are raised, the Biological Safety Supervisor and the University Biological Safety Officer can be contacted for advice and guidance. As well as this, biosafety resources can be located on the Newcastle University Safety Office page to provide further information on a range of safety topics including approved biological agents, biological safety cabinets explanation etc. Our team mainly works in laboratories in the Centre of Bacterial Cell Biology (CBCB) which is a BioSafety Level (BSL) rating 2. Due to lack of space, we also use the laboratories in the Ridley Building which is a BSL rating 1.

PEALS

Within the university, there is a Policy, Ethics and Life Sciences (PEALS) Centre, they aim to "produce world class research focused on the social and ethical debates around the contemporary life sciences. Working with a wide range of academic and community-based partners, we will develop rigorous and socially engaged analysis, innovative teaching, and public dialogue that informs and contributes to future practices and policies." We had a discussion with the executive director, Professor Janice McLaughlin, regarding biosafety and responsibility. To see more about the ethical considerations raised please see our ethics page.

Training

The team undertook a two week period of lab training to minimize risks and accidents during our summer project in the lab. During these two weeks, we familiarized ourselves with basic techniques such as pipetting and the use of machinery (centrifuges, vortexes, nanodrop, microscopes etc.). We also learnt about how the lab is laid out and the waste management procedures specific to the lab. During the training, key safety guidelines were enforced which can be seen in more detail here. Before we entered the lab for training, we read and signed the Control of Substances Hazardous to Health (COSHH) forms after they had been explained by the lab’s Safety Officer Kate Brown.

Bacterial Strains

E. coli (DH5a & XL-1Blue)

DH5a and XL-1Blue are both common K-12 derivatives of E. coli that are classed as hazard group 1 as they are not pathogenic to humans, animals, plants and microorganisms. Although these strains rarely pose a threat to healthy individuals because they have very limited survivability in the environment and normal flora prevents colonization in a healthy colon. The risk is dependent on the circumstance and environment with the most common route being absorption or ingestion. Therefore, optimum safety in the lab is important to limit the potential risk of this colonization. To read in more detail please see this page[http://epa.gov/biotech_rule/pubs/fra/fra004.htm].

B. subtilis (168 & BSB1)

B. subtilis is a common bacterium recovered from water, soil, air and decomposing plant residue. It is also able to reside in a wide range of temperatures which may cause a problem as it has been seen to be associated with a variety of plant diseases such as soft rot in garlic cloves and potato tubers. (Kararah et al., 1985; Claus, 1986) Although B. subtilis is very abundant in the environment, it does not seem to have any specialized attachment mechanisms usually found in organisms that colonize humans. It is only known to cause disease in severely immunosuppressed patients. Horizontal gene transfer has been shown to occur in B. subtilis between strains grown together in soil. Although indigenous virulence factor gene is not observed in B. subtilis, it may be possible for the bacterium to acquire these genes from closely related bacterium within the genus. http://epa.gov/biotech_rule/pubs/pdf/fra009.pdf

B. subtilis L-form

L-forms are a strain of B. subtilis that lacks the cell wall. It is osmotically unstable and even if made large scale, the L-forms will not be able to survive in the environment without specific media needed for stability. Therefore, there are limited risks associated.

Bio-Hazards

Ultraviolet (UV) Radiation

Ultraviolet light is a 180-400nm wavelength non-ionizing radiation and the degree of harm depends on the specific wavelength. The most harmful natural type (180-280nm) is filtered out by the earth’s ozone layer. However, man-made sources of UV radiation can be used in the lab such as transilluminators, germicidal lamps and UV lasers. In our project, we used a transilluminator that uses UV light to allow us to visualise DNA separated by electrophoresis on an agarose gel. A transilluminator uses a UVB light source (280-314nm) which can be dangerous with over exposure. Most commonly, exposure affects the skin and cornea of the eyes. This may result in sunburn, premature skin aging, skin cancer and photokeratitis. In order to prevent these effects, the transilluminator is fitted with a protective shield and when in use gloves and a visor must be worn to minimize UV radiation penetrating the skin and eyes.

Isopropyl β-D-1-thiogalactopyranoside (IPTG)

IPTG is used as a substitute for allolactose, a disaccharide inducer of the lac operon. An alternative to allolactose is needed because it is broken down by β-galactosidase, so concentrations do not remain constant during experiments. However IPTG is unaffected by β-galactosidase and is therefore useful in keeping the expression of genes, under the control of the lac operon, constant. The use of IPTG is necessary for our project because one of our BioBricks (Hbsu-xFP Fusion) is under the control of the Pspac promoter which is IPTG induced. Caution must be taken when using IPTG because the substance is a suspected carcinogen therefore the maximum quantity used in the powder form is 10g and in a working stock of 1M, 1ml.

Chloramphenicol

Chloramphenicol is an antibiotic which is affective against many gram-positive and gram-negative bacteria. However, due to drug resistance, its use in developed countries is limited although it is cheap and easily produced. This is why it is still the antibiotic of choice in countries that are less developed. Although use is limited in the medical field, it is commonly used in research for selection purposes. For our project in specific, we have used chloramphenicol to select for colonies where one of our BioBricks (switch brick) has integrated due to the fact that it contains an antibiotic resistance marker for chloramphenicol. Chloramphenicol may be carcinogenic and cause heritable genetic damage therefore precautions must be taken when working with it. This includes, having the maximum stock solution concentration of 10mg/ml and a maximum quantity of 5g so damage is limited.

Ethanol

Ethanol is commonly used for cleaning purposes and sterilization in the lab to wipe down surfaces and pipettes. The main problem associated with ethanol is irritation especially in the eyes. It may cause pain or permanent corneal damage. Severe systemic effects from inhalation include nausea, headaches and dizziness. In order to minimize the chance of these health hazards the standard concentration for cleaning purposes is 70%. In case of accidental spillage, eye washes are situated by multiple sinks in the lab.

MiniPrep Reagents

Miniprep is a technique regularly used in our project to purify plasmid DNA. It requires a number of hazardous substances including Tris, hydrochloric acid, EDTA, sodium hydroxide, sodium dodecyl sulphate, acetic acid, isopropanol and ethanol. All of the reagents pose a health hazard if inhaled, ingested or absorbed and can target skin, eyes, respiratory tract and mucous membranes. The areas affected depend on the route of exposure. Isopropanol, if inhaled, may cause drowsiness and dizziness. In order to prevent these health risks, the user must wear a lab coat, gloves and goggles. If contact with the skin is made, wash immediately.

References

Claus, D.a.R.C.W.B. (1986) 'Genus Bacillus Cohn 1872', Manual of Systematic Bacteriology, 2(In: P.H.A. Sneath, et al. (eds.), Bergey's), pp. 1105-1139. Kararah, M.A., F.M. Barakat, M.S. Mikhail and Fouly., H.M. (1985) 'Pathophysiology in garlic cloves inoculated with Bacillus subtilis, Bacillus pumilus, and Erwinia carotovora.', Egyptian J. Phytopathol, 17(2), pp. 131-140.