Since 2008, at least seven teams have attempted to use deoxyribonucleases to eliminate the genome of their reprogrammed machine, in order to destroy both the GMOs and their DNA and thereby prevent possible horizontal gene transfer. However, since nucleases are very toxic for cells, many essays were unsuccessuful. Using an arabinose-inducible promoter that could be tightly repressed by glucose, our team successfully cloned in Escherichia coli three distinct nucleases from different phages : protein A1 coming from bacteriophage T5 (BBa_K3027000), Gp3 produced by phage T7 (BBa_K3027001), and YqcG (BBa_K3027002), part of a toxin-antitoxin system encoded in a prophage of Bacillus subtilis. Within our project we have shown that these nucleases present different characteristics regarding the kinetic of DNA elimination and the impact on cell morphology. This collection of nucleases enables the efficient generation of DNA-less bacteria and thus, self-destroying chassis that will be useful for containment purposes. Moreover, we characterized within our DNA-less chassis the activity of a previous biobrick encoding a pollutant degradation pathway.
Our new biobricks :
Proteins that cleave DNA.
- A1 nuclease : BBa_K3027000 and BBa_K3027004 (composite biobrick)
- Gp3 nuclease : BBa_K3027001
- YqcG nuclease : BBa_K3027002
Biobrick whose activity kinetics we have characterized and whose effectiveness was evaluated in a DNA-less chassis :