Safety -

In terms of biological safety, the project ideas of the TU Munich Team 20132 do not raise any more issues than those which have to be considered in every biotechnological work involving genetics and microbiology. However, since the long-term goal of the project ist an application in the environment, the possible effects of the transgenic plants we intend to produce must be considered. Safety is one of the main focuses of our PhyscoFilter project. This includes biosafety, biosecurity and safety in the lab.

BILD/SCHEMA

Biosafety

According to the WHO, biosafety is the prevention of unintentional exposure to pathogens and toxins, or their accidental release. In order to minimize the risk of escape of our moss, we have taken several measures, including a light-triggered killswitch and the use of a non-sporulating Physcomitrella strain. Additionally, we have evaluated every single part we are using in terms of safety.

Issues that we have considered during our research include:

• horizontal gene transfer (EreB)
• vertical gene transfer
• escape

risks concering the team members, 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. It is also not harmful to the environment, because none of transgenes introduced into the moss will increase the vitality of the moss in nature. Furthermore, none of the transgenic plants that we have created is able to produce a toxic substance. The enzymes secreted by our moss for biodegradation (Laccase, EreB, Catechol-2,3-dioxygenase) are not harmful themselves. However, degradation products of hormones, antibiotics and other pollutants could be potentially toxic. Therefore, the reaction products will be tightly monitored. 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.

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 ecological niche for the transgenic moss by just covering an area with an appropriate filter foil. Other ecological ninches can’t be occupied by transgenic moss equipped with our kill switch. BILD mit Schema

Non-sporulating Physcomitrella strains

In order to prevent our moss from spreading in the environment, we looked for strains which are not able to reproduce. 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. The moss life cycle can be affected by phytohormones but also by metabolic processes. At the International Moss Stock Center, there are several Physcomitrella strains available, which are not able to produce spores that can germinate. A possible way to inhibit successful germination is to knock out the enzyme sulfite reductase 1 (SiR1). This protein reduces sulfite to sulfide and is involved in sulfur metabolism. 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.

Safety evaluation for used BioBricks and composite parts

None of the biobricks we produced this year are harmful to humans or the environment. Some enzymes, for example Laccase are already used in the food industry (Quelle). Since we are also using parts of mammalian origin and from S2-organisms, we have filled out the extended safety form, which can be found 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.

pdf/tabelle eingebettet mit allen Biobricks und sicherheitsstufe der organismen

pdf/tabelle mit S2/mammalian organismen und erklärungen

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 (listed here) that we have created could be used to cause harm.

Labsafety

The lab we work in is classified as BSL 1 (biosafety level 1), according to the Union Directive 2000/54/EG and the German "Gesetz zur Regelung der Gentechnik (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 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 the responsible for lab 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.

[Edens et al., 1984]

1. [Edens et al., 1984] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. Cell, 37(2):629–33.