Team:GO Paris-Saclay/Demonstrate
Controlled expression of nuclease genes in bacteria leads to DNA-less cells
Our project aimed at showing the potential of a cell without DNA. The first step was to choose nucleases and characterize what happens to cells producing these nucleases.
We chose 3 nucleases coming from phages: nuclease_A1 (BBa_K3027000) from bacteriophage T5, nuclease_gp3 (BBa_K3027001) from bacteriophage T7 and nuclease_yqcG (BBa_K3027002)(a toxin gene associated with phage-related sequences in Bacillus subtilis genome). Using an arabinose-inducible promoter that could be tightly repressed by glucose, our team successfully cloned the nuclease genes in Escherichia coli. First, we tested the impact of their expression on bacterial growth and survival. To induce nuclease gene expression, we added arabinose to the cultures. As a negative control, we used bacteria carrying the empty vector pBAD24-MoClo.
Figure 1:
Impact of nuclease expression on bacterial growth
Figure 2:
Impact of nuclease gene expression on bacterial survival
Figure 1 shows that arabinose addition led to bacterial growth arrest within 30 min for cells expressing nuclease_A1 (BBa_K3027000) and nuclease_gp3 (BBa_K3027001) but that it took longer (90 min) to observe growth arrest with nuclease_yqcG (BBa_K3027002). In parallel, bacteria were plated to assess survival (Figure 2). Within 30 minutes of arabinose addition, we recovered 1000-fold less bacteria carrying nuclease_A1 (BBa_K3027000) and nuclease_gp3 (BBa_K3027001) than cells carrying the empty vector control. The impact of nuclease_yqcG expression on killing was delayed: a 10-fold decrease in CFU recovery was observed after 90 minutes induction. Taken together, it appeared that, depending on the nuclease gene tested, between 1 and 0.1 % of bacteria are able to survive after 150 minutes induction.
Next, we tested whether induction of nuclease gene expression leads to DNA degradation. In our first experiment we forgot to include ribonuclease during the purification of genomic DNA and therefore recovered both genomic DNA and ribosomal RNA. Figure 3A shows that when we induced expression of nuclease_A1 (BBa_K3027000) and nuclease_gp3 (BBa_K3027001) for 30 min or more, we could no longer recover genomic DNA but ribosomal RNA was still present.
Figure 3:
Impact of nuclease gene expression on bacterial genomic DNA recovery
When we repeated the experiment, we included ribonuclease during the purification. Figure 3B shows that expression of nuclease_yqcG (BBa_K3027001) leads to a slower digestion of genomic DNA than nuclease_A1 (BBa_K3027000) consistent with the corresponding survival kinetics. Taken together, our results show that controlled expression of all three nucleases leads to reduced recovery of genomic bacterial DNA.
Finally, we investigated whether the induction of nuclease gene expression maintains cell integrity for several hours. Following fixation and DNA labelling with DAPI, cells were observed by fluorescence microscopy (Figure 4).
Figure 4:
DNA staining with DAPI after arabinose addition
Most cells expressing nuclease_A1 (BBa_K3027000) and nuclease_gp3 (BBa_K3027001) did not display DAPI-fluorescence 30 minutes after nuclease genes induction (Figure 4), indicating that they have lost DNA. In accordance with previous results, we only observed reduced DAPI staining in cells expressing nuclease_yqcG for 90 min (rather than 30 min). Most of our experiments were carried out for 150 min: for all nucleases tested, we observed that the DNA-less cells conserved their rod-shape for at least two hours. In some time-lapse microscopy performed with cells expressing nuclease_A1, we observed that cells maintained membrane integrity for at least 6 hours. Taken together, our results show that expression of nuclease genes in E. coli leads to DNA-free cells.
Pre-programmed DNA-less cells can be an efficient “cleaning factories”
As a next step in functionalizing our DNA-free-cells, we tested whether cells producing one of the nucleases along with methotrexate-degrading enzymes were able to decrease the concentration of this toxic anti-cancer drug. We wanted to bio-transform a toxic molecule, methotrexate (MTX) with DNA-less cells as such cells cannot proliferate once they have lost their genetic material and are thus a safer chassis.
The Biobrick CPG2 (BBa_K2688003) has been previously shown to bio-transform the anticancer drug MTX into glutamate and DAMPA that are both less toxic molecules.
E. coli BW25113 cells were transformed with plasmid pSB1C3-cpg2 (BBa_K2688003) and pBAD-nuclease_gp3 (BBa_K3027001). We expressed cpg2 gene for 1 hour then we induced the expression of nuclease_gp3 (BBa_K3027001) gene for 30 minutes to degrade DNA, as we have shown that this time lapse is sufficient to degrade DNA. Thereafter, 100 µM of MTX were added to the culture medium containing cells and incubated at 37°C. After 3 hours, samples were collected. Bacterial cells were recovered by filtration and submitted to HPLC analysis to assess the quantity of MTX and DAMPA.
Figure 5:
Methotrexate (MTX) biotransformation into DAMPA by a DNA-less chassis. Cells were incubated 3 hours in LB medium containing 100µM of MTX. LB media without bacteria containing MTX or DAMPA was used as a control. Results were normalized on the percentage of MTX control condition. The final OD600nm of each cultures was determined for each condition.
We observed that bacteria expressing both cpg2 and nuclease_gp3 (BBa_K3027001) were at least as efficient as DNA-proficient cells expressing CPG2 to remove 100 µM MTX from LB media in 3 h (Figure 5). In conclusion, DNA-less cells can perform metabolic degradation for at least several hours after DNA loss.
This result suggests that our DNA-free cell could be used in bioremediation.
DNA-free cells were reprogrammed into ephemeral “RNA cells”
Another possible application of DNA-free cells is to create an “RNA cell”, in which replicating RNA, rather than DNA, is the support of genetic information. In order to carry out this substitution of DNA with RNA, we first had to eliminate the cellular DNA with the 3 characterized nucleases. As the next step in achieving an RNA cell we needed to inject RNA, replicate it and produce proteins from it. We chose to use a bacteriophage (MS2) whose genetic information is carried by RNA. In this work, we tested whether cells that have lost their DNA could be infected by the MS2 phage and produce new virions, which would result from the replication of RNA and its translation into proteins. If successful, these cells would transiently be akin to “RNA cells”.
Cells produced nuclease_A1 for 15 minutes then MS2 phages were added to proceed to infection. It appeared that MS2 titer soared by more than 4 logs between 90 and 120 minutes, following the very same trend as positive control cells (Figure 6). In addition, while genomic DNA could be recovered in control cells 90 and 120 minutes after addition of phages, no DNA was recovered from strain expressing the nuclease_A1 (BBa_K3027000).
Figure 6:
Comparison of MS2 phage titer after incubation with KeioZ1 F’ pBAD24 positive control cells or KeioZ1 F’ cells harbouring a pBAD24 plasmid which encodes nuclease_A1 (BBa_K3027000)
Taken together, our results suggest that MS2 is able to multiply in cells without DNA. Therefore, for a short amount of time, we generated cells where the replicating genetic information was carried by RNA. Although ephemeral we were able to produce an "RNA cell", resembling a cell that may have existed in the RNA world, before DNA evolved.
Figure 7:
Model of our ephemeral RNA-cell. With a little help from RNA phage MS2, we generated a DNA-free cell where the replicating genetic material is RNA
