To ensure we carried out our project in a safe and responsible manner we made sure we checked all important safety requirements. We implemented the notion of Safe-by-Design, received proper safety training before entering the lab, confirmed that all our biological parts meet ML-I requirements, carried out safe and responsible laboratory practices and made certain we disposed of all biological and chemical matter in the proper way.
Sci-Phi 29 facilitates engineering of a repertoire of bacterial species. It provides a set of genetic parts that can be used to assemble a linear plasmid for expression of genes across bacterial species. We want to emphasize that safety and security are important in every possible case. Here we present four possible scenarios for our platform and how we tackle them with the notion of Safe-by-Design. Each section of the infographic below shows a possible scenario, where our platform is used or misused, and this use can be intended or unintended. Each part of the infograhic contains our recommendations on how to handle the corresponding scenario.
To limit irresponsible usage of the linear plasmid, we propose a usage protocol that delineates conditions and scenarios in which the plasmid can be put to use. We recommend that users of the Sci-Phi 29 toolkit adhere to our protocol to prevent escape of the linear plasmid.
The usage protocol contains the following points of attention:
- Strict adherence to barrier design criteria
- Usage limited to biosafety level 1 laboratories
- Usage limited in mixed cultures of unknown composition
Barrier design criteria
The barrier design criteria prevent danger possibly caused by the construct due to its universality.
- Physical containment is to be enforced: The use of the plasmid should be limited to closed systems. Bacteria harboring the linear plasmid must only be cultivated in confined and controlled environments. Products of processes using the linear plasmid must be devoid of bacteria and any waste generated must be treated appropriately (in accordance with proper disposal practices) before disposal. To avoid disrupting ecosystems, usage of the plasmid is not allowed in environmental contexts, outside of the lab and without adequate research having been conducted beforehand.
- Dependence on T7 RNA polymerase: To ensure additional safety, we recommend that the individual components of the plasmid should be expressed under a T7 promoter (or a variant thereof). In such a case, initial expression (kickstarting) of the orthogonal replication is needed. Kickstarting would entail the expression of T7 RNA polymerase under the influence of a promoter specific to the bacteria of interest. Such a design choice separates the bacteria specific promoter from the construct and necessitates the use of an external ‘kickstarter’ plasmid (devoid of an origin of replication, for transient expression of T7 RNA polymerase). The kickstarter plasmid can be changed to increase specificity of initiation and therefore increase safety against introduction into unwanted contaminating organisms.
Biosafety level 1 laboratories
Biosafety level 1 laboratories are authorized to work with well-characterized or studied chemicals and organisms that cause no harm to healthy humans (Laboratory biosafety manual). We therefore recommend that usage of our toolkit is limited to biosafety level 1 organisms. Although this could greatly limit applications of our system, we believe our system is still in the early phase of development and despite careful safety designs and considerations, our plasmid could provide an unintended biological advantage to pathogenic or infectious microbes. Furthermore, we discourage the use of our system in human cells.
Limited usage in mixed cultures of unknown composition
The use of our plasmid system should be avoided in microbial communities of unknown composition. Limiting the usage in microbial communities will prevent dangers associated with selection of pathogenic or infectious organisms in culture.
The laboratory work of our project is carried out in ML-1 lab workspaces at the Bionanoscience Department of the Faculty of Applied Sciences of our university, Delft University of Technology. ML-1 laboratories, comparable to BSL-1 workspaces, are considered as the lowest level of microbiology laboratory biosecurity. Only the use of non-pathogenic microorganisms is allowed. However, we still have strict rules for ML-1 workspaces. Before we were allowed to enter the lab, all team members had to pass the mandatory safety tests to ensure we had knowledge of the safety rules and emergency operation procedures.
These safety tests consisted of the following:
- General building safety (meeting points, emergency numbers, evacuation plans, etc.)
- General laboratory safety (chemicals, waste disposal, clothing, safety precautions, etc.)
- Biological safety (ML-1 grade safety, biological waste, safety precautions, etc.)
- Laser safety (general safety precautions)
On top of these tests, we received lab training and instructions on how to work safely in the Bionanoscience Department, where to discard different types of waste, and how to minimize contamination risks within and outside of the lab. Furthermore, we had to write a research plan and safety proposal. This document had to be approved by the wet lab and safety coordinators of the Bionanoscience Department before we were allowed to start any laboratory work, and was updated throughout our project. The safety proposal can be found here. All our research adheres to the biotechnology regulations of biosafety of The Netherlands.
Lab Safety Rules ﹀
- Lab coats are always mandatory in every laboratory and hallway in an ML-1 zone.
- Short pants, skirts or open shoes are not allowed when working in a laboratory .
- Eating and drinking is not allowed in the laboratory.
- The use of mobile phones is not allowed in the laboratory, except in case of an emergency.
- Personal items (e.g. bags and jackets) are not allowed in the laboratory.
- Hands should be washed on entering and leaving the laboratory.
- Researchers should work clean and tidy.
- The bench should be cleaned with 70% ethanol before the start and after the end of an experiment.
- Researchers should know where to find personal protective equipment (e.g. hand gloves, safety glasses, eye shower) and know how to use them.
- Researchers should know how to use a fume hood.
- Chemicals are stored according to Publicatiereeks Gevaarlijke Stoffen (PGS) 15.
- Chemical waste is disposed of according to the different waste categories.
- Cryogenic liquids are used according to the right procedure.
- Samples are labelled with name of experimentalist, substance, date of production, expiration date and permit number, if applicable.
- Before working with organisms and cell lines you are obliged to make a risk evaluation in the Biosafety module in Lab Servant (Online laboratory safety suite).
- A researcher is responsible for an up-to-date registration.
- It is not allowed to work with any biological material without permission of the Department Wet Lab Coordinator (VM) and/or Biological Safety Officer of the Bionanoscience Department.
- The lab must be clean and tidy.
- The workbench is cleaned with alcohol before and after handling organisms or cells.
- A researcher should take of his/her lab coats before washing his/her hands and when leaving the ML-1 lab.
- Long hair should be done up. (Head)scarves and similar garments should be tucked in the lab coat.
- Jewelry on hands and wrists is not allowed; these are extreme infection sources.
Sci-Phi 29 Safety
General safety is important to work responsibly. But besides general safety, there are safety aspects which specifically apply to our project.
To show independence of hosts of our platform, multiple constructs have been tested across multiple
bacterial species. All strains used for these purposes are non-infectious, non-pathogenic and
scaled under biosafety level 1.
Table 1: Overview of Organisms Used During Our Project ﹀
Host species Host strain Biosafety level Escherichia coli Top 10 1 BL21(DE3) 1 BL21(DE3) pLysS 1 Vibrio natriegens Vmax 1 ATCC14048 1 Pseudomonas putida MC4-5222 1 Bacillus subtilis 110NA 1 168 1
Our part collection provides an array of modular cloning (MoClo) compatible parts. That is why we cloned our constructs in the MoClo backbones from the MoClo Toolkit (Weber et al., 2011). These backbones contain type-specific overhangs. Furthermore, our constructs for Bacillus subtilis were cloned into the MK969 backbone. Finally, expression in Vibrio natriegens was achieved by the use of the pET325Cm-YFP plasmid. All vectors used are derived from backbones which can be used in ML-1 laboratories in accordance to the biotechnology regulations of biosafety for The Netherlands.
Table 2: Overview of Vectors Used for Cloning and Expression ﹀
Vector name Backbone Source pICH41233 pUC19-derived MoClo Toolkit pICH41246 pUC19-derived MoClo Toolkit pICH41308 pUC19-derived MoClo Toolkit pICH41276 pUC19-derived MoClo Toolkit pICH47732 pUC19-derived MoClo Toolkit pICH47742 pUC19-derived MoClo Toolkit pICH47751 pUC19-derived MoClo Toolkit pICH47761 pUC19-derived MoClo Toolkit pICH47802 pUC19-derived MoClo Toolkit pICH47811 pUC19-derived MoClo Toolkit pICH47822 pUC19-derived MoClo Toolkit pAGM8031 pUC19-derived MoClo Toolkit pAGM8055 pUC19-derived MoClo Toolkit pICH50881 pUC19-derived MoClo Toolkit pICH50900 pUC19-derived MoClo Toolkit pICH54022 pUC19-derived MoClo Toolkit ICH54022 pUC19-derived MoClo Toolkit MK969 pAK Addgene pET325Cm-YGFP pET-28 a (+) derived SGI-DNA
Our part collection provides an array of modular cloning (MoClo) compatible parts that can be used for predictable expression of genes across different bacterial species. Our collection consists of these inserts or combinations thereof. To ensure the safety of our project we made sure all of these inserts are safe to work with in ML-1 laboratories.
Table 3: Overview of inserts used to construct our part collection ﹀
Insert Name Derived From Source Part Type 0.1 T7 Promoter T7 phage IDT Promoter 0.5 T7 Promoter T7 phage IDT Promoter WT T7 Promoter T7 phage IDT Promoter T7 sp1 Promoter T7 phage IDT Promoter 0.5 T7 sp1 Promoter T7 phage IDT Promoter T7 sp2 Promoter T7 phage IDT Promoter Pbhr Promoter Synthetic* IDT Promoter Pbhr sp1 Promoter Synthetic* IDT Promoter Pbhr sp2 Promoter Synthetic* IDT Promoter Universal RBS Synthetic** IDT RBS B0032 Synthetic IDT RBS Sarj B0032 Synthetic** IDT RBS Sarj uRBS Synthetic** IDT RBS RiboJ uRBS Synthetic** IDT RBS RiboJ uRBS Synthetic** IDT RBS Φ29 DNA Polymerase (p2) Bacillus phage phi29 IDT CDS Φ29 Terminal Proteins (p3) Bacillus phage phi29 IDT CDS Φ29 Single Stranded Binding Protein (p5) Bacillus phage phi29 IDT CDS Φ29 Double Stranded Binding Protein (p6) Bacillus phage phi29 IDT CDS eGFP Aequorea victoria IDT CDS Harmonised GFP Aequorea victoria IDT CDS TALEsp1 Synthetic* IDT CDS TALEsp2 Synthetic* IDT CDS WT T7 Terminator T7 phage IDT Terminator T7 v7 Terminator T7 phage IDT Terminator
* Yang, S., Liu, Q., Zhang, Y., Du, G., Chen, J., & Kang, Z. (2018). Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology. ACS Synthetic Biology, 7(1), 287–291. https://doi.org/10.1021/acssynbio.7b00258
** Segall-Shapiro, T. H., Sontag, E. D., & Voigt, C. A. (2018). Engineered promoters enable constant gene expression at any copy number in bacteria. Nature Biotechnology, 36(4), 352–358. https://doi.org/10.1038/nbt.4111
Not only biological safety, but also chemical safety is an important factor to consider. During our project we worked with carcinogenic chemicals like SYBR Safe and ethidium bromide. In order to safely work with these chemicals, possible risks were evaluated prior to working with them, together with the biosafety officer of the Bionanoscience Department. To safely work with these chemicals, appropriate measures were taken. Every piece of equipment that comes in touch with these chemicals is located in a contained, marked off area, where gloves are used at all times. When excising bands from an agarose gel, under illumination of UV light, protective glasses were worn besides the standard safety measures.
To complete the circle of safe laboratory practices, we made sure all biological and chemical components are disposed in a responsible and safe manner. These practices apply to make sure that all biological material is contained even after experiments are done. Furthermore, chemicals that are used should never be a risk for anybody outside of the lab.
One of the important aspects of working in an ML-1 lab is the regulation of biological disposal. Genetically modified organisms are not allowed to be released outside the lab and have to be handled with responsibility. All biological waste has been collected in labelled ML-1 waste bottles which were sterilized by autoclaving. Consumables contaminated with biological waste were disposed in ML-1 waste bins which were also sterilized by autoclaving.
The disposal of hazardous chemical waste was carried out according to the protocol of the Bionanoscience Department. For example, we separated SYBR Safe (carcinogenic) waste in a special tank, which was collected by a team of specialists in disposal of hazardous chemical laboratory waste. Contaminated consumables were also disposed in hazardous chemical waste bins.