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.

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 [http://www.moss-stock-center.org/| 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. [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.

Biosecurity

Biosecurity is the prevention of loss, theft, misuse, diversion or intentional release of pathogens and toxins.

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 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.

http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984

1. http://www.ncbi.nlm.nih.gov/pubmed/6327079 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.