Team:Potsdam Bioware/Lab/Safety

Safety assessment

General procedure

In Germany, the work with genetically modified organisms is regulated by:

• the ‘Law on Genetic Engineering’ (-> Gesetz zur Regelung der Gentechnik, GenTG)

• and amendments of the ”Gentechnik-Sicherheitsverordnung (GenTSV)”,
• the “Gentechnik-Verfahrensverordnung (GenTVfV)” and
• the “Gentechnik-Aufzeichnungsverordnung (GenTAufzV)”.

Potsdam Bioware Project 2012

Our laboratory is registered as a Biosafety Level 1 (BSL 1) laboratory according to the ”Gentechnik-Sicherheitsverordnung (GenTSV)”.
Furthermore we had to estimate the project for the appropriate authorities and proof that all organisms and plasmids are classified as BSL 1:
organisms:
E. coli XL1-Blue
Level: 1 Federal Office for Consumer Protection and Food Safety
The XL1-Blue strain allows blue-white color screening for recombinant plasmids and is an excellent host strain for routine cloning applications using plasmid or lambda vectors. XL1-Blue Genotype: recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F´ proAB lacIqZΔM15 Tn10 (Tetr)]. (Genes listed signify mutant alleles. Genes on the F´ episome, however, are wild-type unless indicated otherwise). XL-1 cells are tetracycline resistant. XL1-Blue cells are endonuclease (endA) deficient, which greatly improves the quality of miniprep DNA, and are recombination (recA) deficient, improving insert stability. The hsdR mutation prevents the cleavage of cloned DNA by the EcoK endonuclease system. The lacIqZΔM15 gene on the F´ episome allows blue-white color screening.

CHO cell line
Level 1 Federal Office for Consumer Protection and Food Safety
Cell line: CHO-K1 DSMZ no.: ACC 110; Species: Chinese hamster (Cricetulus griseus); Cell type: ovary cells Origin: subclone from parental CHO cell line that was initiated from an ovary biopsy of an adult Chinese hamster in 1957.

HEK 293 cell line (AAV-HEK Zellen)
Level 1 Federal Office for Consumer Protection and Food Safety
We recommend preparing adeno-associated recombinant virus stocks using the AAV-293 cell line provided. The AAV-293 cells provided are derived from the commonly used HEK293 cell line, but produce higher viral titers. HEK293 cells are human embryonic kidney cells that have been transformed by sheared adenovirus type 5 DNA.2 AAV-293 cells, like HEK293 cells, produce E1 in trans, allowing the production of infectious virus particles when cells are transfected with E1-deleted adenovirus vectors or when co-transfected with the three AAV Helper-Free System plasmids (an ITR-containing plasmid, pAAV-RC, and pHelper).

HT1080 cell line (AAV-HT1080)
ATCC® Number: CCL-121™ Stratagene recommends titering adeno-associated recombinant virus stocks using the AAV-HT1080 cells, a human fibrosarcoma cell line. Stratagene has found the AAV-HT1080 cells to be more permissible to AAV infection than other cells lines, such as 293 cells, and thus able to give more accurate viral titers. In addition, AAV titering protocols using AAV-HT1080 cells have been optimized and are provided in Stratagene’s AAV-Helper Free System Instruction Manual (Catalog #240071).

Adeno-associated Virus
Level 1 Federal Office for Consumer Protection and Food Safety
The Biological Safety Level (BSL) classifications for AAVs are:

• Adeno-associated Virus 2, 3 and 5 have to be handled under BSL 1.
  
• Adeno-associated Virus 1, 4, 6, 7, 8, 9, 10 and 11 have to be handled under BSL 2.
  

This classification was developed based on the fact that only serotypes 2, 3 and 5 are isolated from humans and that their harmlessness has been confirmed in clinical studies. This general classification has to be revised when the virus contains gene sequences with a transforming potential.

Viral vector systems include a vector plasmid that only contains the viral Inverted Terminal Repeats (ITRs) and the genes for Rep and Cap in trans (as in the case of our project). So our virus can be classified as BSL 1 if the following conditions are fulfilled:

• The viral particles do not contain AAV derived sequences other than the ITRs
  
• The viral particles do not contain nucleotide sequences with a risk potential
  

Due to the fact that we work with AAV-2 and we fulfill the conditions stated above, it can be handled under BSL1.

In our project we work with E.coli strains: XL-1 Blue and ER2738, CHO (Chinese Hamster Ovary) cells, HT1080 (Human fibrosarcoma) cells, HEK293 (Human Embryonic Kidney) cells, rAAV (recombinant Adeno-associated virus) and phages VCSM13. These organisms are classified as BSL 1 organisms according to the classification of Federal Office for Consumer Protection and Food Safety (Bundesamt für Verbraucherschutz und Lebensmittelsicherheit)

E. coli ER2738
Level: 1 Federal Office for Consumer Protection and Food Safety E. coli with plasmid for F-pillus, used for phage display.

phages VCSM13 Level: 1 Federal Office for Consumer Protection and Food Safety Safety issues concerning phages:
Even though the phage type that we used is commonly used in laboratories, it requires an extra working bench. Phages are robust organisms that infect pilli bacteria. Therefore, they are not pathogenic for humans and do not cause any risk for people. Nevertheless, while working with phages one has to be careful not to get any phage contamination to other bacterial strains present in the lab.

Safety questions:

1. Would any of your project ideas raise safety issues concerning researchers, the public or the environment?

The current project and further project ideas of the Potsdam Bioware Team 2012 are classified as biosafety level one (S1) work. None of the ideas do raise additional safety issues to the public, the environment or researchers. We comply with safety regulations given by the law and its amendments, including but not limited to handling and storage of genetically modified organisms and the documentation of the work.

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

No, none of our generated BioBrick parts or devices will raise any safety issues concerning the public or the environment.

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

The University of Potsdam supervises biological and chemical laboratories and works according to federal and state regulations. A local biosafety officer has been appointed for the lab and the project. About twice a year, the laboratory is physically inspected by the state Authority for Biotechnological Laboratories to ensure compliance with the law and regulations. In Germany, the work with genetically modified organisms is regulated by the ‘Law on Genetic Engineering’ (Gesetz zur Regelung der Gentechnik, GenTG) and amendments of the ”Gentechnik-Sicherheitsverordnung (GenTS)”, the “Gentechnik-Verfahrensverordnung (GenTVfV)” and the “Gentechnik-Aufzeichnungsverordnung (GenTAufzV)”.

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?

With the beginning widespread use of synthetic genes, devices and systems the standard safety classification based on originating and accepting organism is becoming less appropriate. A more abstracted view of BioBricks with respect to their function and potential interference at the molecular, cellular and organismic level is advised. Known as well as potential risks could be listed in a specific section for each BioBrick in the Partsregistry. In addition to the safety assessment, each iGEM group could give a short assessment of the technology impact based on intended and, if applicable, unintended use.