Team:Bielefeld-Germany/Safety

• Start
• Team
  • Members
  • Photos
  • Bielefeld University
  • Acknowledgements
  • Contact
  • 
• Project
  • Description
  • Achievements
  • Approach
  • Model
  • Database
  • Background
  • Labjournal
  • Protocols
  • Outlook
  • 
• Results
  • Summary
  • Datapage
  • Laccases
  • Immobilization
  • Substrate Analysis
  • Cellulose Binding Domain
  • Shuttle Vector
  • Collaboration
  • 
• Since Regionals
  • Summary
  • Labjournal
  • 
• Human Practice
  • Overview
  • Expert
  • Politics
  • Industry
  • Outreach
  • Education
  • Environment
  • Press
  • 
• Safety
• Judging
• Partner

1. Would any of your project ideas raise safety issues in terms of

• researcher safety,
• public safety, or
• environmental safety?

the listed organisms we use (or from which we use parts) are all part of the basic biosafety level 1:

• Pichia pastoris WT X33 ^[3]
• Pichia pastoris GS115 ^[3]
• Aequeora victoria ^[1]
• Cellulomonas fimi ATCC 484 ^[2]
• Clostridium cellulovorans ^[1]
• Escherichia coli BL21 (DE3)
• Thermus thermophilus HB27 ^[1]
• Bacillus halodurans C-125 ^[1]
• Bacillus pumilus DSM 27 (ATCC7061) ^[1]
• Xanthomonas campestris pv. campestris B100 ^[1]
• Streptomyces lavendulae REN-7
• Streptomyces griseus IFO 13350 ^[3]
• Arabidopsis thaliana Col-0
• Trametes versicolor ^[1]

For assuring safety of the researchers every team member has to take part in special lab safety training. This training includes theoretical and practical parts where the team members learn how to handle (harmful) chemicals, rules for handling the setups and general safety regulations which have to be considered in the lab.

Further, everyone is required to be informed in detail about the properties of every used chemical reagent. This implies that everyone has to read the R/S phrases and consider the recommended safety regulations.

To protect the researcher every person in the lab is required to wear safety clothes which implies long pants, closed shoes, a lab coat, safety glasses and when necessary gloves.

Other students and people without the authority and training do not have access to our iGEM lab. We take care that no organisms which are biological modified leave the lab. In the case that samples, chemicals or something else need to be transferred to another lab in order to continue an experiment we use special containers. For example, solvents are carried in baskets and samples in closed containers. Our project does not include the work with infectious organisms. Cell material can be collected in glas bottles, which are going to be autoclaved or they can be disposed in a special sink, which is connected to a thermical disinfection system from [http://www.envirodts.de/ EnviroDTS].

Figure 1: process scheme of the thermical desinfection system in the lab

In our project we have to work with harmful chemicals. To avoid exposure we have to take special care, for example using gloves or breathing masks if necessary. The most harmful chemicals are listed below with the material safety data sheet ([http://www.chemdb.de/ chemdb.de]):

• [http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=DE&language=de&productNumber=46067&brand=SIGMA Ethidium bromide] for staining the DNA-gel
• [http://www.alfa-ebsoft.com/msds/pdf/german/L15075.PDF Acrylamide] in the SDS-PAGE
• [http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=DE&language=de&productNumber=08253&brand=FLUKA Copper chloride] in the culture medium for the fermentation

To characterize our produced enzymes we chose different substrates. Among the substrate are some harmful compounds:

• [http://de.wikipedia.org/wiki/Estradiol Estradiol]: toxic
• [http://de.wikipedia.org/wiki/Estron Estrone]: toxic
• [http://de.wikipedia.org/wiki/Ethinylestradiol Ethinylestradiol]: toxic
• [http://de.wikipedia.org/wiki/Diclofenac Diclofenac]: toxic
• [http://de.wikipedia.org/wiki/Ibuprofen Ibuprofen]: harmful to health
• [http://de.wikipedia.org/wiki/Naproxen Naproxen]: harmful to health
• [http://de.wikipedia.org/wiki/Naphthalin Naphthalene]: harmful to health and dangerous to environment
• [http://de.wikipedia.org/wiki/Acenaphthen Acenaphthene]: dangerous to environment
• [http://de.wikipedia.org/wiki/Phenanthren Phenanthrene]: dangerous to environment
• [http://de.wikipedia.org/wiki/Anthracen Anthracene]: irritant and dangerous to environment
• [http://de.wikipedia.org/wiki/Lindan Lindane]: toxic and dangerous to environment

These chemicals are stored in the lab regarding the safety regulations. Therefore, solvents are stored in explosion protected lockers and toxic chemicals are locked separately. Of course all chemicals are labeled adequate. Work with harmful chemicals, especially the handling of solvents, is done under a fume hood. Process steps which require the protection from contaminations are carried out under a clean bench.

Some experiments are carried out in special areas in the lab. One example is ethidium bromide which we use for staining our gels. We only use the chemical in a special marked area of our lab and have separate equipment to prevent exposure.

As already mentioned above, we also care about the environment. Therefore, all chemicals are disposed regarding their properties such as toxicity and explosiveness. All chemicals and chemically contaminated trash is disposed by the chemical disposal department. The department is also able to recycle solvents, such as ethanol and acetone (requires only a low grade of contamination). In this case we can get the distilled solvents, labeled as technical solvents, back and can use them for experiments or applications where the pureness is not a crucial factor.

In a future application our laccases could be detached from the immobilization material for example if the CBD (cellulose binding domain) is not strong enough. If they are washed out into the drinking water there is the possibility of public exposure. But usage of laccases in the food industry ^[4] suggest that there is no further issue with the consumption of laccases.

The laccases are known to have a broad spectrum of activity. A possibility is that laccases degrade estrogen or other chemical compounds to toxic degradation products. To exclude the chance of toxic degradation products the laccases are characterized with a broad range of substrates and the degradation products are analyzed.

In summary, we take care about the safety of ourselves, try to work as clean as possible and take care to cause no harm to the environment. The rules and safety measures we consider are standard at the Bielefeld University as well as in Germany.

2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes,

• did you document these issues in the Registry?
• how did you manage to handle the safety issue?
• How could other teams learn from your experience?

No, none of the new BioBrick parts that we constucted this year raise any safety issues. Although we are working with a plant pathogenous organism (Xanthomonas campestris pv. Campestris B100), but we are using a harmless enzyme laccase. Moreover the enzyme laccase of Trametes versicolor is intended for use in the food-processing industry, for example for bakery products and drinks. Furthermore the organisms we used to isolate laccases and our production strain Escherichia coli KRX are S1 organisms, which are not able to survive outside the lab conditions. This is a requirement for S1 organisms ([http://www.bvl.bund.de/SharedDocs/Downloads/06_Gentechnik/register_datenbanken/organismenliste.html?nn=1484542 list of these organisms]) defined by the german act of genetics ([http://www.gesetze-im-internet.de/gentsv/index.html#BJNR023400990BJNE001503320 GenTSV]):

Biosecurity stage 1 Organisms

• are not human-, phyto- or animalpathogen
• do not contain or release organisms containing to a higher risk stage
• are approved by experiments or long term evaluation or do not proliferate in the enviroment because of biological implanted boundaries.

3. Is there a local biosafety group, committee, or review board at your institution?

• If yes, what does your local biosafety group think about your project?
• If no, which specific biosafety rules or guidelines do you have to consider in your country?

There is no Biosafety Committee at our university, only a safety officer. Nevertheless, we have an iGEM instructor, Dr. Jörn Kalinowski, who is kept informed about the project progress the entire time and is responsible for the biosafety of our project. Also, our university has a number of teams for waste management and fire safety.

The general rule at our university is that before every lab course every student has to take part in specific safety training for the course. Further, once a year, depending on the field of study and research, every member of the labs is required to take a refreshment course in lab safety, bio safety, fire safety, waste management, handling gas cylinders and working with hazardous materials. All safety classes include a presentation and a practical part. For example in fire safety every student practices how to use the different types of fire distinguisher.

Before we (the iGEM team) started our lab work we took part at a general lab safety training course. Also, we had specific training for working with genetically modified organisms (GMOs) which is regulated in Germany by a number of laws. The work with GMOs has to be documented carefully. Handling, decontamination and disposal of GMOs is strictly regulated. According to the guidelines genetically modified organisms and contaminated materials can leave the lab after decontamination. We make sure that all of our work is done considering the relevant rules and regulations.

The [http://www.bvl.bund.de/DE/06_Gentechnik/gentechnik_node.html Federal office of consumer protection and safety] (Federal office of consumer protection and safety in [http://www.bvl.bund.de/EN/06_Genetic_Engineering/genetic_engineering_node.html English]) is the institution responsible for biosafety at universities, institutions and so on... All laws, regulations and guidelines can be found here:

• [http://www.bvl.bund.de/DE/06_Gentechnik/03_Antragsteller/08_Rechtsvorschriften/01_Deutschland/gentechnik_rechtsgrundlagen_national_node.html National German laws and regulations]
• [http://www.bvl.bund.de/DE/06_Gentechnik/03_Antragsteller/08_Rechtsvorschriften/02_Europa/gentechnik_rechtsgrundlagen_eu_node.html European regulations and guidelines]
• [http://www.bvl.bund.de/DE/06_Gentechnik/03_Antragsteller/08_Rechtsvorschriften/03_International/gentechnik_internationale_uebereinkuenfte_node.html International contracts and agreements]

The Federal office of consumer protection and safety appoints a team of experts ([http://www.bvl.bund.de/DE/06_Gentechnik/02_Verbraucher/05_Institutionen_fuer_biologische_Sicherheit/02_ZKBS/gentechnik_zkbs_node.html ZKBS], Zentrale Kommission für die Biologische Sicherheit) who determine the risks of GMOs for humans, animals and the environment. Based on their results they can suggest rules and regulations for handling GMOs.

ZKBS in [http://www.bvl.bund.de/EN/06_Genetic_Engineering/ZKBS/01_Allg_Stellungnahmen/genetic_engineering_zkbs_allgStellungnahmen_node.html English]

4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

During brainstorming to realize our project an idea raised to build a symbiosis reactor. we imagined that both organisms could not survive without each other outside the reactor. This idea could pose a greater biosafety in the lab for the researcher himself, for the public and the environment.

Further, everyone should be aware that we are not able to estimate the effects of releasing genetically modified organisms in the environment. That is why we work with a cell free system. Probably, it would be much easier to immobilize the organism itself directly in the filter but we decided to isolate the enzymes due to environmental safety.

References

[http://www.google.de/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CCoQFjAA&url=http%3A%2F%2Fwww.opbw.org%2Fnat_imp%2Fleg_reg%2Fgerm%2FRegulations_Biol_Agents_bacteria.pdf&ei=jOY8UMasLarV4QThu4BQ&usg=AFQjCNFcxERwLdjdjNK8IdHyTs5a-FXjcg [1]] Einstufung von Bakterien in Risikogruppen, Bundesarbeitsblatt 10/2002

[http://www.cect.org/english/vbac.php?cect=4283&lan=en [2]] COLECCIÓN ESPAÑOLA DE CULTIVOS TIPO, www.cect.org/english/vbac.php?cect=4283&lan=en, on www.cect.org, Aug 30, 2012

[http://www.bvl.bund.de/SharedDocs/Downloads/06_Gentechnik/register_datenbanken/organismenliste.pdf;jsessionid=BBE29883867AB94FF1B68820F342520C.1_cid103?__blob=publicationFile&v=4 [3]] Bundesamt für Verbraucherschutz und Lebensmittelsicherheit, www.bvl.bund.de/DE/06_Gentechnik/03_Antragsteller/06_Institutionen_fuer_biologische_Sicherheit/01_ZKBS/03_Organismenliste/gentechnik_zkbs_organismenliste_node, on www.bvl.bund.de, Sep 5, 2012

[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2963825/ [4]] Johann F. Osma, José L. Toca-Herrera, Susana Rodriguez-Couto (2010), Uses of Laccases in the Food Industry, Enzyme Research 2010:1-8