yqcG is a nuclease coming from a prophage of Bacillus subtilis. We have demonstrated that this biobrick can eliminate genomic DNA and thus be used to efficiently generate DNA-less chassis. To characterize the nuclease, its CDS has been inserted in a plasmid under the control of an inducible promoter. This plasmid was then used to transform bacteria which will express the nuclease for the study.
Materials and methods
pBAD24 is a plasmid that contains an ampicillin resistance gene, a pBR322_origin for replication, an araC gene coding the AraC protein and a PBAD promoter. The nuclease_yqcG gene was inserted in the pBAD24 plasmid under the control of PBAD. AraC regulates the expression of genes controlled by the PBAD. The presence of glucose in media inhibits the adenyl cyclase producing cAMP. cAMP forms a complex with the CRP protein to activate the transcription of araC gene. Therefore, when glucose is absent from media, synthesis of AraC is stimulated. This protein forms a homodimer, which represses the transcription of genes under the control of the PBAD promoter, in the absence of arabinose. Arabinose modifies the conformation of the homodimer AraC. This alteration activates the transcription of genes controlled by PBAD. This design allows a tight regulation of the inserted gene by glucose (repression) and arabinose (induction). We modified plasmid pBAD24 to introduce two BsaI sites downstream the PBAD promoter to facilitate cloning using the Golden Gate technique. This new plasmid, named pBAD24-MoClo, allows type IIs cloning with the prefix AATG and the suffix GCTT. This cloning strategy was used to clone nuclease_yqcG gene, yielding the plasmid pBAD24-nuclease_yqcG.
The E. coli KeioZ1F’ strain was transformed with pBAD24-nuclease_yqcG. The resulting strain was characterized as followed:
The strain KeioZ1F’ + pBAD24-nuclease_yqcG was inoculated in 3 ml of LB supplemented with ampicillin (100 μg/mL) and glucose (0.2 %) and grown overnight at 37°C with shaking. The next day, the culture was diluted in LB supplemented with ampicillin in order to have an optical density at 600 nm (O.D.600) equal to 0.1. The culture was shaken at 37°C until O.D.600 0.4. The nuclease was then induced by adding arabinose (0.2 %) to the media at time point t=0. O.D.600 was measured and the cell number was determined by spotting 10 µL of serial dilutions on agar plate (LB + ampicillin + glucose) at different time points : -30 min (i.e. 30 min prior to arabinose induction), +30 min (i.e. 30 min after arabinose induction), +90min, +150 min and “Day after”. This protocol was repeated several times during the summer to have a large amount of data. The negative control uses the strain KeioZ1F’ transformed with pBAD24-MoClo. In this plasmid, no nuclease gene has been inserted.
Result 1: Expression of nuclease gene yqcG inhibits bacterial growth and survival
Addition of arabinose to the growth media results in growth arrest for the strain with the plasmid carrying yqcG 90 minutes after induction (Figure 2). However, the OD600 keeps increasing for the strain with the control plasmid pBAD24-MoClo (without the nuclease gene). Thus, we observed that expression of the yqcG nuclease gene stopped bacterial growth up to 150 minutes upon arabinose induction.
Addition of arabinose to the culture does not affect cell viability of the strain with the control plasmid pBAD24-MoClo. In contrast, the viability of a strain with pBAD24-nuclease_yqcG decreases upon nuclease induction by arabinose (Figure 3). The number of viable bacteria decreases 100-fold, from 3.106 CFU/mL to 3.104 CFU/mL between 30 minutes and 150min of induction. Therefore, yqcG nuclease expression swept away up to 99% of the bacteria.
Result 2: Reduced genomic DNA recovery upon expression of nuclease gene yqcG
The evolution of genomic DNA (gDNA) over time was studied. Following addition of arabinose, one mL of culture was sampled at different times to extract gDNA thanks to a Macherey Nagel kit. The control is the KeioZ1F’ strain carrying the empty vector pBAD24-Moclo.
Genomic DNA is still present after nuclease induction in bacteria with plasmid pBAD24-Moclo. For strains carrying the plasmid pBAD24-nuclease_yqcG, gDNA cannot be recovered after 90 minutes of nuclease induction with arabinose.
Result 3: Reduced DNA staining with DAPI in cells expressing nuclease gene yqcG
To visualize whether cells expressing yqcG were still intact and whether they contained DNA that could be stained with DAPI, a microscopy study was performed using cell fixation and DAPI staining. Following arabinose addition, culture aliquots were taken at different time points (t = 0min, t = +30min, t = + 90min and t = +150 min). Cells were fixed and coloured according to the protocol « Fixation of cells and coloration with DAPI ». The control strain is the KeioZ1F’ strain carrying the empty vector pBAD24-Moclo
Arabinose addition does not affect the DNA staining of strain carrying pBAD24-Moclo (Figure 5A). However, for those containing pBAD24-nuclease_yqcG, the addition of arabinose resulted in the DNA destruction in most of the bacteria after 90 min (Figure 5B), consistent with the reduced gDNA recovery observed at the same time point (Figure 4). Some bacteria still have their DNA intact. Compared to the results obtained with the other nuclease biobricks (INSERT a LINK to the Biobrick A1 and Biobrick gp3), DNA “wipe-out” by yqcG is a relatively slow process as it requires 90 min rather than 30 min induction.
Result 4: Some bacterial cells survive following the expression of nuclease gene yqcG
When bacteria were incubated overnight with arabinose, some cells were able to survive and multiply, as witnessed by the 10-fold survivor increase in CFU recovery between 150 min and overnight incubation (Figure 7). Therefore, some bacteria (or “cheaters”) did survive the nuclease induction. One surviving colony from one of the induction experiments was named “Survivor 1”. We tested whether this strain changed phenotype compared to the original strain that was always maintained in medium with glucose.
Unexpectedly, upon addition of arabinose, growth of “Survivor 1” was stopped (Figure 8) and the survival rate dropped (Figure 9). This experiment was performed only once and here, the drop-in survival was more efficient and faster than for the original clone carrying yqcG. This survivor was lucky to survive the first nuclease induction as yqcG appears to be still fully functional.
Result summary and perspective
Taken together, our results indicate that we can generate DNA-free cells thanks to the controlled expression of nuclease_yqcG in E. coli. However, resistant mutants are able to arise through the insertion of mobile genetic elements in the coding sequence.