About our lab

Material used:
We use open benches and Laminar flow hood / biosafety cabinet with open front to handle biological materials.
We handle cancerogenous materials such as ethidium bromide or polyacrylamide under separate closable hoods. Also acidic and basic solutions are handeled behind closable hoods. All radioactive materials or methods are located in a separate laboratory in the basement.
We also have a separate room for RNA work.

Protection:
For protecting ourselves while doing lab chores, we use full face shields during cutting out gels or any work that involves longer exposure to UV radiation. We certainly use nitril gloves for handling any ethidium bromide and polyacrylamide contaminated materials. Safety glasses are frequently worn, especially if dangerous materials like acids, bases and liquid nitrogene are used or if there is anything that can splash like at the AKTA. Fur using -80°C freezers we use isolated gloves.
The laboratory safety training requirements of the Technical University Berlin can be found here or here (BG RCI).

All Team members received a safety training.

Disposal:
We collect biological waste in big containers within the lab and autoclave it later. The autoclaved waste is then taken care of by the institute. Contaminated and non-contaminated broken glas are collected separately. There is also a separate waste for cancerogenous and environmental hazardous waste like ethidium bromide gels etc.

Biosafety guidelines of the Technical University Berlin:
The Institutional Biosafety Officer (BSO) is responsible for ensuring that laboratories are working safetly with biohazardous materials. This includes the periodic inspection of laboratories, developing emergency plans for handling spills and accidents and investigating laboratory accidents involving biohazardous materials. The BSO also provides technical consultation for researchers on conducting risk assessments, safety practices, and security. Environmental Health and Safety provides services necessary for biological research. These services include biomedical waste disposal, autoclave quality assurance, biosafety cabinet certification, biosafety inspections, training and emergency response. Know the hazards associated with the biological materials and procedures used in the laboratory. Follow approved lab procedures and safety guidelines. Know the emergency procedures. Complete all required training before conducting any lab activity. Utilize all required Personal Protective Equipment. Do not work alone in the laboratory.

German regulations that govern biosafety in research laboratories:
http://www.gmo-safety.eu/
http://www.gmo-safety.eu/links.html
http://ec.europa.eu/food/food/biotechnology/gmfood/qanda_en.pdf
http://www.baua.de/nn_15226/de/Themen-von-A-Z/Biologische-Arbeitsstoffe/TRBA/pdf/TRBA-466.pdf

Identified possible risks
of our project

At this point we want to show that we have dealt with the possible risks of our project.
Risks to the safety and health of team members, or other people working in the lab:
We are working with E.coli, the strains we are using are characterized as non-pathogenic, BSL 1. However, we try to modify E.coli by knocking out genes - therefore, every person who is working in the lab has to take care of the possible risks. Consequently, it is mandatory to wear labcoats and gloves to minimize any risk of contamination. Also for other labwork e.g. dealing with chemicals such as Ethidium bromide, team members have to use the Personal Protective Equipment (PPE) and have to be personally instructed.
Risks to the safety and health of the general public (if any biological materials escaped from our lab):
We are using non-pathogenic E. coli strains and even our genetically modified E.coli strains are not changing the risk of pathogenic influences on human beings. Nevertheless, we are taking care to avoid any possibility of contaminating the environment with our E.coli strain or any kind of non-natural agents.
Risks to the environment (from waste disposal, or from materials escaping from our lab):
Material which was in contact with any organisms is autoclaved by normal BSL-1 procedures. Organic solvents, solutions as well as agarose gels and gloves containing Ethidium bromide are separately disposed according to the guidelines of our institution. We are strictly trying to reduce the amount of contaminated material and also the amount of consumables which are made of plastic.
Risks to security through malicious mis-use by individuals, groups, or countries:
None. The entry of the BSL-1 are of our building is restricted and continuously controlled by security personnel. As our project does not create any E. coli strains or BioBricks with harmful or toxic properties, malicious mis-use by other individuals is ruled out.
What new risks might arise from our project's growth?
If all of our dreams became true, one possible application of our project would be gut cancer treatment. The strains we are using are E.coli. This microorganism is ubiquitous in the human gut-system. Nobody is capable to say what would happen when our modified strains are in fact applied to a human being. It could for instance occur that the applied strain is interacting with the natural bacterial strains in the gut-system - this might provoke irritations and lead to stomach/intestinal problems. However, every developed system will change through evolution - so, nobody is capable to say what exactly will happen by application of the genetically modified E.coli. Any application should be under strict supervision and only be done by professional medical personnel.
For our possible application one could use inducible promoters. When you apply the modified E.coli strain the desired effects from BioBrick gene expression are then only induced in presence of a special non-harming chemical substance (e.g. IPTG).

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Addressing the safety of free accessible probiotic E. coli Nissle Strain 1917

E. coli Nissle 1917 is a probiotic strain used for medical treatment of gastroenterological deseases. The genome structure of the probiotic strain was analyzed by Grozdanov et al. (2004) with three different approaches. While the core gene pool of organisms such as E. coli is conserved and required for basic cellular functions the flexible gene pool allows for instance for variation to enable adaptation to special growth conditions. This variability offers a potential of gaining pathogenicity within nonpathogenic (also called commensal) strains as E. coli Nissle. Grozdanov et al. claim that E. coli strain Nissle 1917 is a typical example of a nonpathogenic, commensal E. coli isolate. However, commensal E. coli strains may develop into pathogenic variants and vice versa under certain environmental conditions. Therefore, the evolutionary aspects of the development of traits of colonization and commensalism in particular strains were also studied. Nonpathogenic and pathogenic E. coli strains differ in the presence of genetic information that may contribute to specific virulence traits (virulence factors) or to successful survival and fitness of the bacteria in the host. Both nonpathogenic and pathogenic E. coli strains are able to colonize the gut and are well adapted to the conditions found in the large intestine. Most of the determinants coding for such factors are thought to be acquired by horizontal gene transfer and are often clustered on the chromosome on genomic islands. A genomic island (GEI) is part of a genome that has evidence of horizontal origins.[1]
The results of Grozdanov et al. demonstrate that factors as adhesins, iron uptake systems, and proteases do not necessarily have to be considered virulence-associated factors but can also contribute to the fitness and adaptability of nonpathogenic bacteria.[1]



Thus, many of these gained features by gene transfer can be considered rather as contributing to fitness (e.g., iron uptake systems, bacteriocins, proteases, fimbriae, and other adhesins), thereby generally increasing adaptability, competitiveness, and the ability to efficiently colonize the human body, in contrast to typical virulence factors which are directly involved in infection. This finding is supported by the findings of Grozdanov et al., indicating that DNA regions of GEI INissle 1917 to GEI IVNissle 1917 can also be detected in nonpathogenic E. coli isolates. In addition, GEIs contain multiple copies of highly homologous functional and nonfunctional ORFs of mobile genetic elements which do not directly contribute to virulence.[1]

The specific combination of characteristic features might mirror the process of evolutionary withdrawal from the pathogenic E. coli serotype O6 lineages, indicating that horizontal gene transfer, subsequent loss of horizontally acquired genetic information, and point mutations have been involved in the evolution of strain Nissle 1917.[1]

In comparing the genome content of one nonpathogenic, probiotic E. coli O6 strain with that of two UPEC (urinary pathogenic E. coli) O6 isolates, it becomes clear that it is difficult to define true virulence factors. Taken together, it depends on the niche or growth conditions (a nonpathogenic milieu such as the gastrointestinal tract versus a pathogenic milieu such as the urinary tract) to show whether certain fitness factors can also promote virulence.[1]

Bacteria of the microflora like EcN interact with the lumen of the intestine by adhering factors and biofilm formation. These organisms facilitate the exchange of nutrients and induction of the hosts immunity. The interaction between intestinal flora and the host’s epithelial cells can besides genetic predisposition along with a modern diet lead to increased energy storage causing pathogenic implications for the host. Since the cause of pathogenic E. coli strains are virulence factors, these factors can promote adaptability of the strain and hence provide a broad array of diseases.[2]

In experiments of Dr. Schmidt, EcN itself appeared to be invasive in the chosen cell culture model. The propagated safety of the strain is therefore not ensured. Dr. Schmitdt concluded that it is necessary to investigate the traits of EcN closer to minimize the risk for patients consuming EcN. Simultaneously a genetical modificaion of EcN by deletion of the mobile genetic elements can contribute to the genomes stability. This way the safety of the strain with respect to modifications of its genome, thus leading to an increase in probiotic traits, can be provided so that employment of transport vehicles for in situ expression of therapeutic molecules is also made possible for seriously ill patients. Furthermore it was shown that EcN in artificial systems such as cell cultures or gnotobiotic mice exhibit a pathogenic potential.[2]

All in all, Whether a commensal E. coli will develop into a pathogen depends not only on the acquisition of fitness-conferring genetic information enabling successful colonization of the host, but also on the presence of functional genes directly contributing to pathogenesis. [1]

References:
[1] L. Grozdanov, C. Raasch, J. Schulze, U. Sonnenborn, G. Gottschalk, J. Hacker, U. Dobrindt, Analysis of the Genome Structure of the Nonpathogenic Probiotic Escherichia coli Strain Nissle 1917, ASM, 2004, 186, 5432-5441.

[2] D. S. Schmidt, Dissertation, Technischen Universität Dresden, 2009.

Further Literature:

The WHO website: http://www.who.int/topics/biosafety/en/
Especially: http://www.who.int/csr/resources/publications/HSE_GAR_BDP_2010_2/en/
http://www.who.int/ihr/publications/who_hse_ihr_2012.12/en/
http://www.who.int/ihr/publications/biosafety/en/

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