Team:TU-Munich/Project/Safety

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Biosafety

Biosafety deals with the prevention of unintented exposure to pathogens and toxins, or their accidental release http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf WHO.

Figure 1: The three main parts of our safety system

Biosafety to us means minimizing the risks concering the team working in the lab, the general public and the environment. Physcomitrella itself does not pose any risks to the health of the researcher or the general public, since it is endemic to many parts of the world. In the lab, we are able to cultivate our moss in bioreactors in which the flow of substances is tightly controlled. They can even be used for the production of therapeutic proteins, as done by the [http://www.greenovation.com/ greenovation] company. For usage in a sewage plant, such reactors could be upscaled and special filter systems could be applied to ensure that no moss can escape into the environment. However, our long-term goal is to use moss filters in polluted environments but at the same time ensuring the highest level of biological safety.

We believe that the main biosafety-issues that have to be addressed regarding our project are

• toxicity of recombinant proteins
• toxicity of degradation products
• transgene pollution (vertical and horizontal gene transfer)

In order to minimize the aforementioned risks, we have taken several measures, including a light-triggered killswitch, non-sporulating Physcomitrella strains and the evaluation of the genetic parts that we are using in terms of safety.

Toxicity of recombinant proteins

The most obvious safety threat is that the recombinant proteins might be toxic and thus have a negative effect on the environment. This is the foundation of our safety consideration - that the products from all parts and genetic circuits must not cause harm by themselves. All our effector proteins (laccase, catechol-2,3-dioxygenase, erythromycin esterase B) are all from S1 organisms and are not toxic. For example, laccases are already used in the food industry http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2963825/ Osma et al., 2010. For the establishment of a eukaryotic expression system, we are also using parts of mammalian origin and from S2-organisms (see safety forms below). We have made the decisions to use parts from S2 organism carefully and only after consulting our safety officer. He could ensure us that the parts and circuits we are using do not pose any threat. When possible, we have tried to substitute parts from S2 organisms with parts from S1 organisms. All of the parts we are using (such as a polioviral internal ribosome entry site (IRES) and the Ig-Kappa secretion signal from mouse) are widely used in molecular biology laboratories all around the world and are considered safe.

Safety evaluation for used BioBricks and composite parts

Table 1: Extended safety evaluation for biobricks of mammalian origin or from S2 organisms

Table 2: Extended safety evaluation for biobricks of mammalian origin or from S2 organisms

Toxicity of degradation products

None of the transgenic plants that we have created is able to produce a toxic substance. However, degradation products of hormones, antibiotics and other pollutants could be potentially toxic. Therefore, the reaction products will be monitored using LC/MS analysis. For example for EreB, the transformation products of erythromycin possess no endocrine and mutagenic properties http://www.endetech.eu/sites/default/files/field/image/2013.06.07-Oral presentation ICWRER 2013.pdf endetech. In general, it is very difficult to find data on the toxicity of these degradation products.

Additionally, the substances used in bioaccumulation (Glutathion-S-transferase, Proteinphosphatase 1) are not harmful themselves, but moss that has bound toxic substances (e.g. microcystin) in high concentrations is potentially toxic and has to be disposed of separately and with caution.

Transgene pollution

Transgene pollution describes the spread of transgenes beyond the genetically modified species by natural gene flow mechanisms http://www.cabi.org/cabreviews/FullTextPDF/2003/20033177371.pdf Commandeur et al., 2003. Transgenes can spread from transgenic to non-transgenic populations of the same species or related wild species (vertical gene transfer) or to entirely different species such as bacteria (horizontal gene transfer). Because the impact of novel genes on other species is unpredictable, transgene pollution is undesirable and could pose a risk to the ecosystem. When talking about transgenes, one also has to differentiate between primary transgenes such as our effectors and superfluous DNA sequences such as antibiotic resistance markers.

Preventing vertical gene transfer with non-sporulating Physcomitrella strains

Figure 2: Sporophytes of P. patens WT (A) and DSiR1 (B–D) lines 8 weeks after induction of sporulation. The spores of WT plants develop until they are mature in the spore capsules which then tear open and set free the spores. In the DSiR1 lines the spore capsules crack open when the spores inside are still immature, Wiedemann et al., 2010

In order to prevent vertical gene transfer and to limit the spread of the moss in the environment, we looked for strains which are not able to form mature spores. P. patens is monoicous, meaning that male and female organs are produced in one plant. Normally, at the tips of adult gametophores the sexual organs, antheridia (male) and archegonia (female), are produced under inducing conditions. After fertilisation of the egg inside the archegonium a sporophyte develops which contains approx. 5000 spores. A possible way to inhibit successful germination of these spores is to knock out the enzyme sulfite reductase 1 (SiR1). This protein reduces sulfite to sulfide and is involved in sulfur metabolism. http://www.sciencedirect.com/science/article/pii/S0014579310002462 Wiedemann et al., 2010 disrupted PpSiR1 by homologous recombination and found that ΔSiR1 plants showed strong developmental alterations and are unable to produce mature spores. In the ΔSiR1 lines, only one third of the number of sporophytes was formed, the spore capsules cracked open when the spores inside were still immature and these mutant spores did not germinate. Since it takes more time to establish a mature culture with the knock-out mutants and considering the limited time in iGEM competitions, we did not actually use this mutant in our experiments for iGEM. However, the mutant can easily be ordered from the [http://www.moss-stock-center.org/| International Moss Stock Center]. A consideration for the future would be to design the integration vector in a way that targeted gene knockouts in the disulfite reductase gene are possible, thus inhibiting sporulation and having a targeted integration of constructs.

Horizontal gene transfer

Concerning horizontal gene transfer, the main safety threat would be the spread of transgenes from our moss to bacteria. The transfer of primary transgenes or marker genes could lead to new bacteria strains that express these genes and could pass them on to pathogenic strains. But while this process is very common in bacteria, apart from agrobacterium tumefaciens, there are few examples of natural gene transfer between bacteria and higher eukaryotes http://www.cabi.org/cabreviews/FullTextPDF/2003/20033177371.pdf Commandeur et al., 2003.

For the transgene to remain in the bacterial population and thus in the environment, there has to be a strong selective pressure. This could be the case in highly contaminated environments, where the ability to break down a toxic pollutant is beneficial. We are aware that our moss carries transgenes that could be advantageous to bacteria living in these polluted environments. But since all of the effectors that we are testing actually come from bacteria, we do no think that this is the main problem. The genes for these effectors are already present in the bacteria population. Nevertheless, we need to have a closer look at antibiotic resistance genes such as ereB and the selection marker nptII. Antibiotic resistance genes are the focus of attention because of their potentially strong and general selective advantage in human pathogens. But in the case of ereB, which comes from E.coli, the resistance gene is already present in the bacterial population on plasmids while in the moss it is controlled by a moss promotor and integrated in the moss genome. Therefore, it is much more likely that in case of ereB, the horizontal gene transfer occurs between bacteria and not between bacteria and moss.

A possible way to eliminate superfluous DNA sequences such as the nptII marker after a successful transformation is the use of a two-component site-specific recombination system such as Cre-loxP http://www.ncbi.nlm.nih.gov/pubmed/1660141 Dale and Ow, 1991. The recombinase Cre recognizes short sequences known (loxP) and is then able to excise DNA betweeen two loxP sites that are in the same orientation. Therefore, marker genes flanked by loxP sites can be efficiently excised from transgenic plants if Cre is present.

Light-triggered Killswitch

In order to prevent our moss from escaping into the environment, we have included a light-triggered killswitch. When exposed to red-light, a nuclease system is activated which destroys all genetic material, killing the moss and preventing the spread of genetic material of the moss. With this genetic circuit it would be possible to create an niche for the transgenic moss by just covering an area with a blue filter foil. For more information, look at the killswitch page.

Biosecurity

Biosecurity is the prevention of loss, theft, misuse, diversion or intentional release of pathogens and toxins. Physcomitrella itself is in no way pathogenic and is endemic to many parts of the world. However, just like with every transgenic organism, there is the theoretical possibility to use Physcomitrella to cause harm, e.g. when it is used to secrete toxic substances. Nevertheless, other organisms which have for example shorter generation times or are pathogenic by nature seem to be more appropriate for such dual-use applications. Additionally, none of the transgenic plants we created could be used to cause harm.

Labsafety

The lab we work in is classified as BSL 1 (biosafety level 1), according to the [http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32000L0054:en:NOT|European Union Directive 2000/54/EG] and the German "Gesetz zur Regelung der Gentechnik ([http://www.gesetze-im-internet.de/gentg/| GenTG])" (law for the regulation of genetic engineering). There is a total of four Biosafety levels, with BSL 1 being the lowest and BSL 4 being the highest. This classification of the respective Biosafety levels is very similar to the one given in the [http://www.who.int/csr/resources/publications/biosafety/en/Biosafety7.pdf| World Health Organization (WHO) Laboratory Biosafety Manual]. Work inside a BSL 1 lab, such as ours, involves no devices that are potentially harmful to the researchers if they act according to the general precautionary measures. Especially, no pathogenic organisms are used.

A regular safety briefing and a lecture about the legal basics concerning biotechnology and genetic engineering are basic elements of our education at TU Munich. In this context, the handling of biological material, dangerous aspects of chemicals and the circumstances and protocols at the lab we work in are explained. Additionally a special safety briefing was held for all iGEM students by Dr. Martin Schlapschy, who is responsible for safety in Prof. Skerras lab.

All of us have worked in laboratories before and have experience with biological parts and chemicals. When we are unsure about the safety measures that have to be taken when handling certain chemicals and devices, we always have the support of our instructors and the researchers working at Prof. Skerras lab.

Safety precautions during molecular biology experiments

Just like in every other biochemical laboratory, there are substances and devices in our lab which are potentially dangerous. Here are three examples of how these situations are handled in our lab.

1. In every laboratory of molecular biology, specific chemicals are required for the staining of DNA, in order to make it visible on agarose gels. Most of them directly intercalate into double-stranded DNA, making them carcinogenic. The substance we use is ethidium bromide. To prevent skin contact, we wear protective gloves made from nitrile rubber and change them frequently to prevent contamination. All gels and materials that come into contact with ethidium bromide are disposed of separately. This is done in order to prevent their unintended leakage into the environment with subsequent harm to humans, animals and plants.
2. Methods of molecular biology often require strong acids or bases, like hydrochloric acid, or toxic substances such as methanol. We handle them with extreme caution under a fume hood and dispose of them separately.
3. Many devices in the lab can be potentially dangerous towards researchers if they are used carelessly or in the wrong way. One example for this are lamps emitting ultraviolet radiation, which can cause damage to the eyes. When dealing with UV radiation, we always wear safety helmets made out of plexiglas. We are aware of the potential harm caused by devices that we are using and thus can protect ourselves appropriately.

Safety Forms

Basic_Safety_Form.pdf

Safety_Form2.pdf

Safety forms were approved on October 3, 2013 by Evan Appleton.

References

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2982853/ Berends et al., 2010 Nuclease Expression by Staphylococcus aureus Facilitates Escape from Neutrophil Extracellular Traps. J Innate Immun. 2(6): 576–586

http://www.cabi.org/cabreviews/FullTextPDF/2003/20033177371.pdf Commandeur et al., 2003 The biosafety of molecular farming in plants. AgBiotechNet 2003, Vol. 5 April, ABN 110

http://www.ncbi.nlm.nih.gov/pubmed/1660141 Dale and Ow, 1991 Gene transfer with subsequent removal of the selection gene from the host genome. Proc Natl Acad Sci USA. 88(23):10558-62

http://www.ncbi.nlm.nih.gov/pubmed/16026881 Hartenbach & Fussenegger, 2005 Autoregulated, bidirectional and multicistronic gas-inducible mammalian as well as lentiviral expression vectors. J Biotechnol. 120(1): 83-98

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2963825/ Osma et al., 2010 Uses of Laccases in the Food Industry. Enzyme Research, 2010: 918761

http://www.sciencedirect.com/science/article/pii/S0014579310002462 Wiedemann et al, 2010 Targeted knock-out of a gene encoding sulfite reductase in the moss Physcomitrella patens affects gametophytic and sporophytic development. FEBS Letters. 548(11): 2271–2278

http://www.ncbi.nlm.nih.gov/pubmed/22366317 Zakeri et al., 2012 Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A. 109(12): E690-7

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