Team:GO Paris-Saclay/DNAFreeCell
Objective
We have shown that cells without DNA do not lysis immediately after complete degradation of DNA. They persist for up to 6 hours, there are still macromolecules such as ribosomal RNAs and DNA-free cells can perform cellular functions like finishing a cell division. This suggests that cells without DNA are filled with proteins. In addition, our mathematic model predicts that DNA-less cell should still contain a high level of proteins.
CPG2 (BBa_K2688003) has been previously shown to be able to biotransform the anticancer drug methotrexate (MTX) into glutamate and DAMPA, less toxic molecules ( 2018 iGEM GO_Paris-Saclay project ). Of note E. coli K12 strain is resistant to MTX.
We wanted to take advantage of both discoveries. To test the metabolic capacities of a DNA-less bacteria, we evaluated MTX biotransformation by a bacteria producing both nuclease_Gp3 and CPG2. We previously observed that 1 h after incubating bacteria with MTX, the biotransformation of MTX is incomplete ( Kinetic of MTX biotransformation ). We therefore chose to analyze MTX biotransformation after 3 h of incubation.
As a proof of concept, we successively induced the expression of the MTX degradation pathway (BBa_K2688003) and then of nuclease_gp3 (BBa_K3027001). Thereafter bacteria were incubated in the presence of MTX. We monitored MTX biotransformation by HPLC analysis.
Material & methods
E. coli K12 BW25113 cells were transformed with 2 plasmids with different replication origin and antibiotic resistance genes: pBAD-nuclease_gp3 (BBa_K3027001) and pSB1C3-cpg2 (BBa_K2688003). Of note, the expression of cpg2 and nuclease_gp3 are under the control of promoters induced by 0.5 mM IPTG and 0.2 % arabinose, respectively. Strain harbouring only pSB1C3-cpg2 (BBa_K2688003) or pSB1C3-tet (BBa_R0040) were grown in the same conditions and used as positive and negative control, respectively.
The MTX biotransformation assay is illustrated in Figure 1. Briefly, cells were precultured at 37°C overnight under shaking in a LB medium supplemented with glucose (0,2%), ampicillin (100µg/mL) and chloramphenicol (30µg/mL). Thereafter, strains were inoculated at an optical density of 0.1 (OD600) in LB medium containing ampicillin, chloramphenicol and IPTG (0.5mM). Once the OD reaches 0.4, arabinose (0.2%) was added in culture media and cells were kept at 37°C with agitation. Thirty minutes later, cells were diluted at OD600=0.2 in 1mL of LB supplemented with ampicillin, chloramphenicol, IPTG, arabinose and 100mM of MTX were added. Two hundred µl were harvested after 3 h incubation with bacteria. The OD600 was also determined at this time point. Samples were filtered (0.2 µm) and submitted to HPLC analysis performed using a reverse phase C18 column as previously described (see the detailed protocol of HPLC analysis method in the Registry page of BBa_K2688003 - HPLC analysis protocol ). Pure 100 µM MTX and DAMPA were used as controls for HPLC analysis. Results were normalized on the percentage of MTX control condition.
Figure 1: MTX biotransformation assay in DNA-proficient or DNA-less cells
Results:
Figure 2: Methotrexate biotransformation 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 was determined for each condition.MTX biotransformation assay in DNA-proficient or DNA-less cells
Results are presented in Figure 2. After 3 h no MTX has been transformed into DAMPA when incubated in LB medium. The MTX is stable and does not turn spontaneously into DAMPA. The MTX is not converted in DAMPA either when incubated with cells carrying pSB1C3-tet0.
The OD600 of the culture increased from 0.1 to ≈ 3 and 0.45 for DNA-proficient and DNA-less cell populations, respectively.
After 3 h, 87% and 98 % of the MTX was biotransformed into DAMPA by DNA-proficient and DNA-less cells expressing CPG2, respectively. This indicates that DNA-less cells expressing CPG2 can efficiently biotransform MTX.
Moreover DNA-less cells (harboring both pBAD-nuclease_gp3 and pSB1C3-cpg2) seems to have more efficiently biotransformed MTX than DNA-proficient cells (harboring only pSB1C3- cpg2) although the OD600 is almost 8 times lower in DNA-less than in DNA-proficient population of cells.
Discussion & conclusions
The nuclease_gp3 gene was induced 30 min before incubating bacteria with MTX. We previously shown by DNA-DAPI-labelling-microscopy that in such condition, DNA is completely removed from the cells . Moreover, the analysis of the final OD600nm measured in each condition confirmed the expression of Gp3 endonuclease since the growth of the E. coli BW25113 strain harbouring a plasmid encoding cpg2 (pSB1C3-cpg2, BBa_K2688003) and pBAD-nuclease_gp3 (BBa_K3027001) had stopped. This indicates that the Gp3 nuclease was produced and degraded DNA.
We observed that bacteria expressing both cpg2 and gp3 endonuclease were at least as efficient as DNA-proficient cells producing CPG2 to remove 100 µM MTX from LB media in 3 h. We can exclude the possibility that this biotransformation was due solely to the activity of « cheaters » that escaped to Gp3 action since we have shown that they represent only a minor fraction of the population (less than 1 %). If DNA-proficient cheaters would have been the only cells able to metabolize MTX, we would have excepted a reduced MTX biotransformation in absence of DNA. This is not the case. On the opposite, our data indicate that MTX biotransformation may be more efficient in absence than in presence of DNA although the final bacterial density is 8 times lower for DNA-less than in DNA-proficient cell populations. This result has to be confirmed by statistical analysis (repeated experiments) but could suggest that stopping cell metabolism through DNA degradation may reallocate energetic resources in favor of MTX biotransformation.
This result demonstrate that DNA-less cells are still metabolically active and can be efficient ‘cleaning factories’. In conclusion, DNA-less cells can perform metabolic degradation for at least several hours after DNA loss. Such cells could be useful for bioremediation. These cells don’t divide and consequently don’t spread DNA-based genetic information.
