Before starting our lab work we took a « Health and Safety » training on Thursday March 4th. Safety equipment and the people responsible for different safety measures were presented to the whole team.
In our project we worked solely with bacteria, strains of E.Coli bacteria that were proven to be non-pathological and have minimal impact on the environment. We use K-12 they are in the Risk Group 1. It contains non-pathogenic organisms and we work in a laboratory which class in Biosafety Level 1. By taking precautions presented during our Health and Safety training, we minimised the risk of bacterial leaks and contamination. In the eventual case of a leak we planned to use amoxicilline, nitrofurantoïne or triméthoprime antibiotics and we made sure to work with plasmids that did not contain resistance to any of the aforesaid antibiotics.
We planned to create a new part, a sorbitol promoter. We chose a part originating from a Low Risk bacterial Parent and we ordered it through IDT or TWIST (custom gene synthesis).
These images show how our lab was set up to optimize our safety and the safety of our environment.
For each new manipulation, the team asks the laboratory manager how to correctly avoid risks of bacterial contamination and for all additional safety measures required to carry out the experiment. When disposing of our bacteria we put them in a liquid biohazard trash that contains chlorine bleach.
Safety in the design of our project
It is necessary to introduce control mechanisms when working with genetically engineered organisms in the context of synthetic biology. To make our parts and constructions safe we developed systems to be put in place to make sure that bacteria containing our designed plasmids autodestruct. There are 2 of these systems:
- The kill switch
The bacteria need carbon sources to survive. We are basing our project on an article(1) that shows there is a natural hierarchy that naturally exists in the K12 MG1655 Escherichia Coli strand. This is in fact the strand that was used in the article
that inspired our project ( reference article ).
The order of promoter activation is the following :
The last promoter would allow the production of a protein such as a toxin that would induce the death of the cell. The ribose promoter being induced by the consumption of ribose, the cell would start to autodestruct when the ribose in the medium would begin to be consumed.
To control our system even more, we can look at the killing switch project that was done by the iGEM Pasteur Paris team in 2018.
Their idea :
When the bacteria would be in an environment outside of the human body, therefore in environments with temperatures below 37°C, the bacteria would overproduce a toxin. The amount of toxin would be so high that it would no longer be taken care of by the antitoxin and this would cause the death of the bacteria.
To apply this to our model :
This could be used in a possible industrial application. When the bacteria have produced the wanted proteins thanks to our tool, the user could reduce the heat to induce cell death and prevent any eventual contamination. It would therefore be possible to control bacterial death in all steps of production
(1)“Hierarchy of non-glucose sugars in Escherichia Coli.” Aidelberg Guy et al, BMC Systems Biology., 2014 , PMID: 25539838
- The toxin/antitoxin system
For the final step of our project, we would have liked to develop a double toxin/antitoxin system.
The final goal of our project was to produce 2 plasmids. We had planned on building the first plasmid for it to contain the gene of the toxin A and the antitoxin B while building the second containing the gene of the toxin B and the gene of the antitoxin A.
This system could have allowed us to prevent plasmid transfer. The bacteria must contain both plasmids to survive, and if it doesn’t the bacteria would die due to a high level of one of the toxins and a lack of antitoxin. We would then be sure of the death of any bacteria that lacks one of the plasmids. This mechansim is called post-segregational killing = PSK.
We can take for example the model that iGEM Toulouse 2016 had started to develop while adapting it to our model, and trying to enhance it for it to be re-used
To take the same example, we will keep the following double toxin/antitoxin system :
While using the composit part :
Plasmid 1 : toxin A = zeta / antitoxin B = mazE
Plasmid 2 : toxin B = epsilon / antitoxin A = mazF
To express the toxin and antitoxin genes we would need to use the constitutive promoter of E.Coli.
We would need to keep an Ribosomal Binding Site (RBS) upstream of each gene. The RBS is a sequence of nucleotides upstream of the start codon of an mRNAtranscript that is responsible for the recruitment of a ribosome during the initiation of protein translation.
The iGEM Toulouse 2016 team had put in place a riboswitch system to block the expression of the toxin during the cloning step. This system would need to be improved because the second plasmid did not pass the cloning stage
- Igem Toulouse 2016
- Régine Brielle. Etude fonctionnelle d’un système toxine-antitoxine de type I exprimé par Staphylococcus aureus et d’ARN régulateurs associés aux ribosomes bactériens. Médecine humaine et pathologie. Université Rennes 1, 2016. Français. NNT : 2016REN1B037.. .tel-01691695
- Mutschler H, Gebhardt M, Shoeman RL, Meinhart A (2011) A Novel Mechanism of Programmed Cell Death in Bacteria by Toxin–Antitoxin Systems Corrupts Peptidoglycan Synthesis. PLoS Biol 9(3): e1001033. doi:10.1371/journal.pbio.1001033
- Topp S. and Gallivan JP (2008). Riboswitches in unexpected places—A synthetic riboswitch in a protein coding region. RNA 14: 2498–2503
Safety in our Human Practices
Our survey on the position of Women in Hard Sciences was done in a way so the responders are anonymous and the answers will be used to carry out a statistical study that will be published in a booklet.
During our interviews all of the subjects are adults and have given their personal authorization to be recorded during the interview. We then proceeded in writing texts containing elements from these interviews. We did not make these texts visible to the public eye without the written agreement of the subjects at hand. By contacting the Office of Judicial Affairs of our University we made sure that the collection and exploitation of this data were aligned with the interior regulations of our institution.
In the context of Human Practices we also organised 2 public events, an escape game and an art exhibition. The escape game took place in a building belonging to the Faculty of Sciences at the Nantes University. Our escape game took place in an old lab room. The floor we were on is used for office-space, however there is a connection to other floors that still contain active labs. For safety measures set by the occupants of the building we could only open our escape game to employees of Nantes University.
Our art exhibition however, took place in a building owned by Nantes University that is permanently open to the public, so there were no restrictions on the entry to our exhibition.