Project Safety Concerns

The safety concerns of our project were a very important issue for this years iGEM Team of the Technische Universität Braunschweig. Because the use of an antibiotic resistance was necessary for selection of bacteria we included an ampicillin resistance gene in our final construct. Ampicillin is a standard antibiotic commonly used in everyday laboratory work. The methane monooxygenase (MMO) plasmid construct for our E. cowli was planned to be expressed via a promoter which is inducible by methane. Although there is a constitutive promoter in the construct first, cloning according to iGEM standard biobrick format allows an easy exchange of parts. Therefore, various variants are easily constructed out of the broad iGEM Registry in case safety concerns arise. Additionally, we prepared special alginate beads in which our E. cowli will be embedded. These special beads allow the diffusion of gases and offer necessary nutritions. Since E. cowli is not able to grow in the natural rumen fluid of the cow, the beads enable its growth, controlling its localisation at once.
For further application we planned to include a kill switch system based on a light induced promoter. Usually the risk of bacteria passing the abomasum of a cow is very low. Nevertheless an additional light induced kill switch will disable E. cowli to live out of its provided rumen system decreasing the risk of an uncontrolled distribution.

Light Induced Kill Switch

In order to further minimize any risk due to the application of our project we are planning to introduce a system for increased security. Since our transformed E. cowli is a genetically modified organism (GMO) and should therefore not be released into the environment without control a kill switch is considered to be useful. A kill switch is based in a specific combination of genes allowing an inducible growth of the cells, but only under defined conditions. Hence, an uncontrolled proliferation of E. cowli could be prevented.

There are already examples for a wide range of possible kill switch systems in the literature which are based on many different combinations of genes. Several genes coding for toxins are commonly used under the control of an inducible promoter. As toxic substances always involve risks and our E. cowli should not be triggered to produce them and thus affect the environment of the cow’s rumen we decided to use an auxotrophy-based kill switch.

To strengthen the spirit of iGEM and to support the extensive iGEM Registry we are planning to introduce the Luminesensor System of the 2012 iGEM Team Peking (https://2012.igem.org/Team:Peking/Project/Luminesensor/Design) to our cells in order to control their growth.

As shown in figure 1 blue light unlocks the N-terminal cap of the VVD domain allowing a dimerization of the VVD domains and subsequently the N-terminal domains (NTD) of the LexA protein which are fused to the N-terminus of the VVD domain. The dimers are able to bind to the promoter of a certain reporter gene inhibiting transcription initiation.

Accordingly, we planned to design an auxotrophy-based kill switch system for our E. cowli cells. Here, the E. coli strain used for the transformation should lack the ability to produce a certain essential amino acid. This auxotrophy should then be complemented by the appropriate gene under the control of the promoter which can be repressed by the NTD dimer. Hence, the growth of E. cowli would be limited to the inside of the cow’s rumen and abolished once the cells are exposed to light. This type of kill switch system would be particularly useful in case E. cowli is accidently released through the cow’s excretions which are subsequently used in biogas plants. Furthermore, no uncontrolled spreading of E. cowli in the environment would have to be feared.

It should be noted that, in order to render this system functional, an E. coli strain incapable of the expression of the LexA protein is needed. Otherwise the inducible promotor would be permanently repressed, inhibiting the expression of the essential amino acid.

References

1. https://2012.igem.org/Team:Peking/Project/Luminesensor/Design
2. Figure 1 from iGEM Team Peking 2012

Laboratory Safety Concerns

Bacterial Strains Used

In order to minimize the working risk as much as possible, the iGEM Team Braunschweig 2014 exclusively worked with non-pathogenic organisms classified as risk group S1.

Strain	Genotype	Risk Group	Risk to Humans
E. coli XL1-Blue-MRF‘	endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 F'[ ::Tn10 proAB+ lacIq Δ(lacZ)M15 Amy CmR] hsdR17(rK- mK+)	S1	Non-pathogenic, may cause irritation to skin, eyes and respiratory tract, may affect kidneys
Methylococcus capsulatus	wildtype	S1	Non-pathogenic, may cause irritation to skin, eyes and respiratory tract, may affect kidneys
E. coli BL21 DE3	F– ompT gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]	S1	Non-pathogenic, may cause irritation to skin, eyes and respiratory tract, may affect kidneys
E. coli JM109	endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+ Δ(lac-proAB) e14- [F' traD36 proAB+ lacIq lacZΔM15] hsdR17(rK-mK+)	S1	Non-pathogenic, may cause irritation to skin, eyes and respiratory tract, may affect kidneys

Safety equipment

The lab was standardly provided with safety equipment according to the S1 safety forms. Safety showers, eye showers, fire-extinguishers and a fire blanket were installed and easily accessible. Furthermore, lab coats, safety gloves and goggles were available at any time in the lab. Specially labelled areas were restricted to working with carcinogenic substances such as ethidium bromide.

Personal safety

Before starting any work in the lab every team member had to be given an instruction concerning health and safety. Additionally there was a tour of our own and adjacent labs with a supervisor. Instructions were given by a supervisor when using new equipment and techniques. Hazardous and carcinogenic chemicals and substances were avoided whenever possible. In case these chemicals could not be substituted, safety equipment such as special gloves were used. Furthermore, to prevent any risk, the team members worked exclusively in groups of at least two members.

Sterile Work

To keep our lab sterile, working spaces and necessary equipment were cleaned with 70 % ethanol. Ethanol is easily flammable and was therefore used carefully and only for small areas. To avoid contaminations of our cultures and for selection antibiotics were added to the used media with the note that they can cause health problems in large amounts. Furthermore, they can cause undesirable resistances in bacteria and thus were used only if necessary. Ethidium bromide was used for gel electrophoresis with special care because of its DNA-intercalating and cancerogenous properties. Use of UV light was necessary for visualization of DNA in agarose gels.

Bioethics

Bioethics was a very important topic for the team. Working with bacteria as chassis for recombinant protein production is common in science and generally accepted in society. Therefore, no bioethical questions were expected to arise in this matter. In case our project is turned into reality and cows are provided with our E. cowli strain we expect a lot of bioethical questions from the public. People are expected to react sceptically due to our planned use of recombinant organisms in animals intended for food production, which, in fact, is reasonable. The work on animals probably has to be handled in a more sensitive way than the work on other, simpler organisms such as bacteria. It has to be questioned whether the advantages for climate change resulting from the use of E. cowli can compensate the risk every recombinant organism brings up in the environment. Alternatively we planned to design a barn filter provided with a methane filter system based on E. cowli. This technology would render it possible to decrease methane emission without providing animals directly with a recombinant organism. Therefore ethical problems are minimised as well.

Environmental Impacts

In case our project will be realized in cows, it is necessary to reconsider every possible risk recombinant organisms bring to nature and environment. Although there is no possible use of our project regarding national issues and although no toxins or pathogenic substances are expressed, it would be important to question as many risks as possible. Under these circumstances E. cowli will be justifiable due to the positive impact on the minimisation of climate change coming along with it. Additionally, construction of a kill switch is planned in order to avoid any possible risk in case E. cowli escapes into the environment. Furthermore, the application of E. cowli as a part of a barn filter system prevents every risk of its integration into the cow's rumen.

Working with Methane

Due to the fact that we had to use methane during the experiments with Methylococcus capsulatus and our transformed E. cowli various security aspects had to be considered. Since methane is a flammable gas which, when mixed with air in a certain ratio, is also explosive we took the following safety precautions:
The setup was exclusively handled with the correct ratio of methane, air and carbon dioxide under a hood without power sockets or other potential sources of electrical sparks or fire. All experiments were conducted at methane-air-ratios under 5% (Lower Explosive or Flammable Limit LEL/LFL) and over 15% (Upper Explosive or Flammable Limit UEL/UFL) to eliminate the risk of a combustible atmosphere. Hence, we accepted suboptimal conditions for the purpose of safety concerns. Gas cylinders were stored according to the manufacturer’s instructions and closed completely when no experiment was running. Before using the respective gas for creating the needed ratio the system pressure of each cylinder was set to 1 bar. All equipment, including flow controllers, tubes and valves were screened for leak tightness regularly and their suitability for the used gas pressures.