Team:LMU-TUM Munich/Safety

Safety

Introduction

Biosafety is defined as "the discipline addressing the safe handling and containment of infectious microorganisms and hazardous biological materials."(1) According to the U.S. Department of Health and Human Services and the CDC.
For our team the security and safety measures of our project has the highest priority. Since there are many inherent risks to the work at the iGem laboratory, that's why we have decided to thoroughly present the dangers that we have analyzed while working at it. Firstly it is relevant to mention that we used non-pathogenic E. Coli strains like BL21 and Xl1-blue for the cloning part of the project, which are typical strains used at the labs. Nevertheless we have had extreme caution to prevent any spreading of used living organisms, since this were S1 genetically modified organisms.
The field of synthetic biology provides us with the possibility to work with living organisms and modify them. There are many safety measures that are very important to consider while investigating and working at the laboratory for this purpose. Since the base of synthetic biology is the work with genetically modified organisms (GMO), it is necessary to look out for risk factors like spreading of the organism or infection from the investigators. It is also important to highlight that molecular biology works at the laboratory involves the use of some harmful chemicals, which is another aspect of the safety to be considered.

Safety at the laboratory

The Laboratory in which the iGem Project has been conducted, at the Chair of biological chemistry is classified as a laboratory with the biosafety level S1. According to the German law of the regulation of GM technology (Gesetz zur Regelung der Gentechnik) there are 4 levels of biosafety, namely from S1 to S4. In the Level S1 there is considered that no risk for human health or the environment is present.(2) order to guarantee the integrity of all the team members and the environment, we took some steps prior to the beginning of our lab work. All members who were going to be performing tasks at the lab were required to attend to a lecture about the risks and precautions while working with GMOs. It was conducted by Dr. Martin Schlapschy in charge of the safety of the laboratory. In this presentation information about the regulation of biology by governmental institutions in Germany and the obligations each laboratory has to prove having a good and ethical practice was also included. After that each team member was given an introduction to the laboratory, in order to get to know it. Factors like disposal of chemicals, GMOs and handling of dangerous chemicals or sensitive cells (cell culture) were specifically emphasized and thoroughly explained.
It is important to underline some important safety measures that have to be taken while conducting molecular biology experiments:

• The use of Ethidium bromide, since it is considered to be a mutagenic chemical compound. The use of gloves, and safety googles was obligatory while using it. (4) This measures also applied for compound like Hydrochloric acid and Ammonia, additionally this were to be handled with the protection of a fume hood, due to the volatility of some of them.
• The use of a special protective mask in order to protect the eyes from the UV Light while analyzing gels.
• The use of certain machines at the laboratory posed some risks as well. As an example the use of big centrifuges, which if mishandled could cause harm to the investigators.
  

The kill-switch

One key factor of guaranteeing the safety of our project and preventing in advance any dangers, is the implementation of a well-known kill-switch. In this case we worked in cooperation with the iGEM-team of Slovenia and Freiburg, who each provided us with their Biobrick (BBa_K404113 - HSV-thymidine kinase fused to mouse guanylate kinase, iGEM Freiburg 2010 and BBa_K782063 - CMV-promoter, iGEM Slovenia 2012). This specific kill switch is known as the Thymidine kinase / ganciclovir system. In order to enhance the current Biobrick, we have repaired a point mutation that led otherwise to a pstI restriction location. An additional Poly-A-tail should also stabilize the mRNA later on. For more details, see our experiment protocol further down.

The debate around bioprinting

The topic of bioprinting, due to the fact of being relatively young, is still currently being discussed in international ethics and safety forums and in courts. This is why we consider it to be essential to briefly present and discuss the main concerns around bioprinting. It is important to mention that there is a broad spectrum of perspectives and disciplines, in which key questions about tissue printing can be analyzed.
The world is facing a lot of challenges nowadays with the constant improvement and advances in the field of biology. These being questions about the ethic of some projects and furthermore the regulation of the reproduction of the same experiments, in the case of its success. This is why it is very important to consider all these political, social and ethical issues bonded to the scope and effect of the project. It is worth mentioning how there is a big debate about organ transplantation already since it first became possible in 1945 when the first kidney was successfully transplanted (HRSA). (4) With bioprinting a lot of new questions around patentability, affordability towards the different social strata, clinical safety and social acceptance arise and are to be discussed and dealt with.
One of the biggest security concerns around the use of 3D printed organs for transplantation is the tort liability of physicians, added to the intellectual property debate of 3D produced organs. Considering the fact that the transplantation of these organs needs still years of research of becoming a reality, the governments and regulation organisms are facing a great challenge in regulating the use and manufacture of 3D printed organs. To illustrate this problem, as an example, the Food and Drug Administration (FDA) is in charge of regulating medical devices as artificial hearts. Meanwhile is Organ Donation regulated by another governmental agreements, like in the case of Illinois, the Anatomical gift act. [http://www.globalforumljd.org/sites/default/files/docs/cop/For%20a%20new%20heart%2C%20just%20click%20print.pdf →to be referenced properly.] Another important factor to consider and discuss is the patentability of such a printer, or in the given case of the printed products. This is where the question comes, whether or not a printed organ should be considered as artificial or normal organs.

Kopiervorlagen

Seitenverantwortliche/r:General (Javier); Killswitch (Enes)

Literaturreferenz

Literaturreferenz

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Textformatierung

kursiv
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Strich

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Links

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Introduction

Implementation of a kill-switch in cells engineered for tissue printing

Inducible termination of synthetic therapeuticals reveals a key improvement in biosafety. For our 3D tissue printing approach we wanted to implement the thymidine kinase/ganciclovir system. Ganciclovir is a prodrug that is toxic for thymidine kinase producing cells, since it efficiently initiates apoptosis. The transfected thymidine kinase is derived by the herpes simplex virus which is why untransfected cells will not be influenced by this system. For this, HSV-thymidine kinase phohsphorylates ganciclovir which is then further phosphorylated by cellular kinases to active ganciclovir-triphosphate. The purine nucleoside analogon is then incorporated into the DNA causing inhibition of DNA synthesis and thus leading to apoptosis [Figure 1]. For our purposes we will use the Biobricks BBa_K404113 (HSV-thymidine kinase fused to mouse guanylate kinase, iGEM Freiburg 2010) and BBa_K782063 (CMV-promoter, iGEM Slovenia 2012). These parts were sequenced and repaired via QuickChange (site-directed mutagenesis) in order to remove a point mutation that led to a pstI restriction site. An additional Poly-A-tail should reveal higher expression efficiency through more stable mRNAs. This was compared to the old Biobrick without the point mutation. Via Light and fluorescence microscopy the cell viability will be monitored and analysed over time.

Applied methods are:

- Cell culture
- Multiwell-based viability testing
- Microscopic assays for viability testing
- FACS-based viability assays

Design

Experiments

Proof of concept

Demonstrate

Discussion

References