Introduction
Safety is a crucial issue for the iGEM projects. As students imagine their own project, they have to make sure that their idea is not hazardous. Likewise, the project's development in the lab is a second challenge for young students in terms of security. Working on living organisms and biological parts requires good laboratory practices to prevent spreading, contamination or physical injuries.
A good laboratory practice
To prevent disorder and accidents, the lab was divided in two parts : a wet laboratory for experiments and a dry laboratory for personal working. Students who were performing experiments were always wearing lab coats and kept experiment materials in the wet laboratory.
Chemical product handling
Students who were experimenting sometimes had to use dangerous chemical products. Extra precautions were taken to handle them:
| Products
|
Dangers
|
Precautions
|
Usages
|
| EtBr (Ethidium Bromide)
|
Mutagenic agent and cancer-causing agent
|
Always manipulate EtBr wearing gloves - Add EtBR in cool solution only to prevent inhalation - Set up a special place and gather material to manipulate EtBr only
|
DNA visualization in agarose gel
|
| Phenol / Chloroform
|
Mutagenic agent, toxic agent, irritating agent and cancer-causing agent
|
Always manipulate Phenol / Chloroform wearing gloves under a hood
|
DNA extraction
|
Contaminated waste was put in special trash, and packaged to be treated elsewhere in a safe manner.
Organisms and Biological parts used in experiments
| Organism
|
Strain
|
Risk group
|
| Escherichia coli
|
DH5a
|
1
|
| Escherichia coli
|
BL21
|
1
|
| Escherichia coli
|
PhB1040 strain 594
|
1
|
Our project was designed to use two different strains of E. coli. These organisms are non-pathogenic chassis ranked in class 1. They do not induce any effects on humans and we could manipulate them safely without any special equipment in BLS 1 (Biosafety Level 1) laboratory.
Biological parts
| Biological part name
|
Origin
|
Use
|
| dCas9 NM
|
DNA synthesis
|
Fused to FRB/FKBP12 system, this part wil allow us to change DNA conformation
|
| dCas9 ST
|
DNA synthesis
|
Fused to FRB/FKBP12 system, this part wil allow us to change DNA conformation
|
| FRB/FKBP12 system
|
|
Fused to dCas9 NM and dCas9 ST, this part wil allow us to change DNA conformation
|
| Tripartite GFP
|
|
Fused to dCas9 NM ans dCas9 ST, this part will confirm that two DNA strands are closed
|
| K1372001
|
Registry part
|
This part produces a tRNA supressor when salicylate is added to the medium
|
Biological parts we engineered were integrated in E. coli. New activities of these engineered bacteria are not dangerous for human health. Especially dcas9 do not have nuclease activity. Besides RNA guide do not target human genes.
Since we generated genetically modified organisms, we had to make sure it was not spread in the environment. We put biological waste in special trashes which were heated at high temperature to kill microorganisms. Then it was treated as usual waste. Moreover dcas9 proteins were not integrated into the genome of sexually reproducing organisms. By this way, we prevent new genes transfert to other organisms than E. coli.
Perspectives of our project and safety issues
Our project aims to develop a new molecular tool to bring closer DNA strands in bacterial cells. If we manage to achieve our project, this new biological tool could only be used by scientists interested in DNA conformation study.
They will be able to target any DNA sequences by designing their own guide RNAs. In order to prevent physical injuries and genetically modified organisms spreading, they will have to take the same precautions we took during the internship listed above.
Conclusion
To conclude our new tool will provide new possibilities in biology research. Scientists will be able to change DNA 3D organization and thus study links between DNA organization and cellular functions in bacteria. As our new tool will used only by scientists it do not really pose new safety issues.
