General lab safety

Fortunately, our university allowed us to enter the lab this year from June to October despite the COVID-19 pandemic. TU Delft actively kept track of the news and followed instructions from health authorities, the National Institute for Public Health and the Environment (RIVM). We followed these instructions carefully.

The wetlab part of our project was performed in the ML-1 area, which is situated at the Bionanoscience department of our Faculty of Applied Sciences on the Delft University of Technology campus. ML-1 is considered to be the lowest biosafety level, corresponding to BSL-1. After passing the mandatory ML1 tests we were allowed to enter and work at two labs.We worked in one regular ML-1 lab and one ML-1 lab meant for bacteriophage work only (Figure 1).

Prior to starting our experiments, all (wetlab) team members passed the obligatory safety tests about:

  • General safety of the Faculty of Applied Sciences
  • General Laboratory safety
  • Biological safety ML-1
  • Laser safety for Laser Users

Below you can find a summary of the rules employees and students at the TU Delft have to follow to guarantee a safe work environment:

In addition to completing the safety tests, we also received lab training on how to safely operate most of the techniques used in basic experiments. We agreed upon rules on how to work safely in our lab to minimize potential risks to laboratory personnel and the environment. We also learned how to discard different types of waste and how to minimize contamination risk. To work in the special bacteriophage lab we received a separate training, as extra safety measures are required to contain the phages and prevent contamination. We learned how to clean phage contaminated surfaces properly and how to correctly perform experiments with phages.
Apart from working safely we also made sure we designed experiments that we could perform safely. We wrote a safety report to evaluate the risks of our experiments including: experimental details, designs, safety regarding COVID-19 and biological safety information. This safety report was approved by Susanne Hage, wetlab coordinator of the department of Bionanoscience of the TU Delft, and Marinka Almering, Biosafety Officer of the Faculty of Applied Sciences of TU Delft. When we made adjustments to our project, we updated the proposal and got it approved before starting the new experiments.

Safe Experimental Design

ML-1 level

We designed all our experiments to comply with the ML-1 safety level. Classification of microorganisms or toxins can differ between countries or regions, we followed the legislation of the TU Delft. Prior to working in the lab we checked that all organisms, vectors and parts are in accordance with the ML-1 level and the Dutch law.


To comply with our ML-1 lab we made sure that all our microorganisms were ML-1. We used E. coli BL21 (DE3) and E. coli DH5 alpha as our chassis organisms. In addition we used E. coli BL21 (DE3) as a host for our phage engineering experiments. We decided to work with the T7 bacteriophage, as it is also classified as ML-1. We exclusively performed phage experiments in a separate bacteriophage lab to prevent unintended E. coli infection (Figure 1).


We made sure to express safe inserts. The Cry7Ca1 toxin is specific to insects, and can be used in an ML-1 lab without separate acceptance from the GMO bureau.
We also expressed YmdB (RNAse II inhibitor), Mini-III (cleaves shRNA), 4-hydroxybenzoate Decarboxylase (degrades phenol) (for more information see Design). These molecules were all not toxic and classified as ML-1. All of these molecules have not been reported to be harmful to humans.


For our research we used different backbones vectors: pKD46, G322 - pUC57 - OriLR - deGFP, pTWIST_bsdBCCD_CO, pSB1C3_BPUL_GA, pTWIST_Cry, pBbB7a - GFP, pBbA2k - RFP, pBbE8c - RFP, pCas9-CR4, pKDsgRNA-p15. Some of these plasmids were kindly provided by other labs from the BN department and some were ordered via Addgene. Material transfer agreement (MTA) regulations were followed according to the rules.


We used several chemicals that are hazardous, these include Ethidium bromide, 2X Laemmli Sample Buffer and SYBR™ Safe. We have only worked with these chemicals in low concentrations and solely worked with these toxins in the designated area. This area was marked with special tape, and we agreed to all work with gloves in that area; these gloves were colored differently compared with the general stock. In addition, any contaminated equipment did not leave this area.


Safety is one of our important design requirements. PHOCUS is a targeted bacteriophage-based biopesticide used to control the desert locust crisis. Our aim is to kill the locusts by producing toxic molecules in their gut. These molecules will be produced by the gut bacteria after infection with our engineered bacteriophage. The toxins we want to produce are the locust specific Cry7Ca1 toxin and short-hairpin RNAs (shRNAs) that target essential locust genes (for more information see Design).
Whilst working on our project, we learned that in biology, nothing is 100% safe. Therefore, we decided to focus on identifying potential risks and having appropriate risk management measures in place to reduce these to an acceptable degree (Figure 1).

Figure 1. Overview of Safe-By-Design measures for the design of PHOCUS.

Field trials

During interviews with experts in the field of, among others, phage biology and toxicology, potential safety concerns were mapped out and discussed. To address these concerns, risk mitigation strategies were developed and lab experiments were designed to test the safety of PHOCUS. We realized that, for PHOCUS to be used in the real world, it first has to go through field trials. To be admissible for these large-scale tests, more safety tests have to be done. Therefore, we investigated which data had to be gathered and designed the experiments accordingly, in addition to studying the corresponding legislation.

From our interview with Dr. Cecile van der Vlugt, senior risk assessor at the Dutch National Institute for Public Health and the Environment, we learned that even though legislation might be absent or non-permissive, the best approach to overcoming noncompliant legislation is by performing a risk assessment of PHOCUS. We most likely have to comply with three types of regulations:

  • GMO regulations: Before taking a GMO into the environment, one needs to make a risk assessment for other organisms in the environment, e.g. we need to prove that the phage will not propagate indefinitely in nature.
  • Insecticide regulations: Every insecticide needs to pass regulations before it can be applied. Answering questions such as; how specific is it? Does it persist in the environment?
  • Food regulations: This will also apply to PHOCUS as it is a ‘living’ insecticide. Regulators will look at toxin/pathogenic effects of the modification made in the phage and how it could be passed to other organisms.

There are many different risks associated with the implementation of PHOCUS for which data should be gathered at a lab scale. This way we can prove the theoretical claims we have previously made, i.e. that PHOCUS is safe to humans. To move PHOCUS beyond the lab, many experiments must be performed to test its:

  • Toxicity to non-target organisms (GM phage, Cry7Ca1, RNAi)
  • Pathogenicity to non-target organisms (GM phage, Cry7Ca1, RNAi)
  • Stability and potential to accumulate (GM phage, Cry7Ca1, RNAi)
  • Uniqueness of sequence targeted (RNAi)
  • Potential gene flow of insert (GM phage)
  • Specificity to locusts (CryCa1, RNAi)

It should be noted that our perception of what is acceptable keeps changing. Therefore the list should be iterated over and altered after continuous discussions with risk assessors and managers. This is extra relevant in relation to our project, as we have a novel application and therefore there may be potentially unidentified risks that could surface over time.

After completion of the lab-scale risk assessment and acceptance by the responsible authorities, PHOCUS can move to field trials. Here, PHOCUS will be subject to different tests in an outside environment, i.e. an environmental risk assessment (ERA) [50]. The experiments performed could be the outside variant of the ones performed in the lab. This data has to be generated again as the actual environment is much more complex and could lead to different interactions. The specifics of what data exactly needs to be gathered remains imprecise. Primarily because our approach is novel and the responsible authorities have not yet drawn up the related legislation. They will likely do this using a case by case approach, as advised by scholars [52, 53]. Still, it will likely remain comparable to existing ERAs for GMO, pesticide and food regulations, e.g. the ERA for GM plants in Australia,which looks at "toxicity, allergenicity, nutritional profile, agronomic characteristics, increased disease burden, spread and persistence of the GMO, gene flow etc." [53]. Further directives for the use of biological control agents are available [54]. While critique of the contemporary ERA on GMOs, which use Cry toxins in transgenic plants, is also presented [55], it becomes clear that experts on risk assessment do not agree on the best approach. Additionally, different countries also have different procedures. Therefore, it is essential to understand that there is not one set way in which PHOCUS will gain approval in all countries, and its regulations should be assessed in close collaboration with the responsible authorities of the affected countries.

After the risk assessments have been conducted, it is important to submit these results to the Pesticide Referee Group (PRG). This is an independent group of scientific experts on pesticides. They assess the data submitted and create a shortlist of pesticides they deem acceptable. This list is specified for its intended use. The FAO advises countries on which pesticides to use. They only buy and advise pesticides that have been shortlisted by the PRG. Lastly, all countries have their own legislative freedom, therefore PHOCUS would still need to be admitted in every country individually.