Team:GO Paris-Saclay/Parts/GP3
Nuclease_gp3 : BBa_K3027001
A protein that cleaves DNA : an endonuclease
Gp3 is a endonuclease coming from bacteriophage T7. 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
The gene encoding nuclease_Gp3 was cloned in the plasmid pBAD24-MoClo, resulting in pBAD24-nuclease_gp3. This plasmid contains an ampicillin resistance gene, the pBR322_origin for replication, an araC gene encoding the AraC transcriptional regulator, the arabinose-inducible PBAD promoter and a RBS upstream of the nuclease gene. 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).
The E. coli KeioZ1F’ strain was transformed with pBAD24-nuclease_gp3. The resulting strain was characterized as followed:
The strain KeioZ1F’ + pBAD24-nuclease_gp3 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 (OD600) equal to 0.1. The culture was shaken at 37°C until OD600=0.4. Expression of nuclease_gp3 was then induced by adding arabinose (0.2 %) to the media at time point t=0. OD600 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 performed on at least five occasions during the summer to have a large amount of data for nuclease_gp3. The negative control in all these experiments was performed with the KeioZ1F’ strain carrying pBAD24-MoClo (this plasmid does not carry the nuclease gene).
Results
Result 1: Expression of nuclease_gp3 inhibits bacterial growth and survival
Addition of arabinose to the growth media resulted in a growth arrest for the strain expressing nuclease_gp3 while the OD600 kept increasing for the strain carrying the control plasmid (pBAD24-MoClo). We observed that the gp3 nuclease expression stopped bacterial growth up to 150 min upon arabinose induction.
Addition of arabinose did not affect cell viability of the strain with the control plasmid (Figure 3). In contrast, the viability of the strain with pBAD24-nuclease_gp3 decreased ca. 100-fold within 30 min of nuclease induction with arabinose (from 7x105 CFU/mL to 8x103 CFU/mL from time -30 min to +30 min after induction). After 150 minutes of induction of nuclease_gp3 we could recover only 0.3 % of the viable bacteria that were present before induction. Therefore, gp3 nuclease expression swept away up to 99.7% of the bacteria.
Result 2: Reduced genomic DNA recovery upon expression of nuclease_gp3
Next, we followed the impact of nuclease induction on bacterial genomic DNA (gDNA). Following addition of arabinose, one mL of culture was sampled at different times to extract gDNA using a Promega kit (cat A1120) following manufacturer’s instruction except that RNase was not added. The KeioZ1F’ strain transformed with pBAD24-MoClo was used as a control.
At all timepoints, gDNA and ribosomal RNA (rRNA) can be recovered from the control bacteria carrying plasmid pBAD24-MoClo. In contrast, as soon as 30 min after arabinose addition we could not recover gDNA from cells carrying pBAD24-nuclease_gp3, while rRNA was present in all samples. Thus, induction of the expression of nuclease_gp3 leads to the destruction of bacterial DNA.
Result 3: Reduced DNA staining with DAPI in cells expressing nuclease_gp3
To visualize whether cells expressing gp3 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 stained 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 to the control cultures did not affect bacterial morphology nor bacterial DNA staining (Figure 5A). However, for bacteria carrying pBAD24-nuclease_gp3, both morphology and DNA staining were impacted (Figure 5B). Even right after arabinose was added to the cells (T=0 min), the bacterial cells looked longer and wider than control cells and not all cells exhibited DNA staining. Within 30 minutes of nuclease induction, only a minority of cells show some staining with DAPI. Therefore, within 30 min of expression of nuclease_gp3, we generated bacterial cells free of DNA.
Result 4: Some bacterial cells survive following the expression of nuclease_gp3
When bacteria were incubated overnight with arabinose, some cells were able to survive and multiply, as witnessed by an increase in OD (Figure 6) and 1000-fold increase in CFU recovery between 150 min and overnight incubation in the presence of arabinose (Figure 7).
Therefore, some bacteria (or “cheaters”) did survive the nuclease induction. We collected three colonies from two independent induction experiments: the strains were called “Survivor 1”, “Survivor 2”, and “Survivor 3”. Next, we tested whether the three “Survivor” strains changed phenotype compared to the original strain that was always maintained in medium with glucose (Figure 8 and 9). Although we only show the results for Survivor 2 and 3, the results were identical for all three survivors: they no longer exhibited slowed growth or decreased survival when arabinose was added to the cells.
In order to understand how these bacteria were now able to escape death, we extracted plasmid DNA from the survivors and introduced it into strain KeioZ1F’. The resulting transformants were resistant to the negative effect of arabinose addition on growth, suggesting that the plasmids from survivors carry mutations disabling the nuclease gene or its expression. Sequence analysis of the plasmids revealed the presence of new DNA sequences inside the CDS of gp3 for Survivor clones 1, 2 and 3. These sequences were analyzed with Blastx.
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Blastx results for the sequence inserted inside the nuclease CDS of survivor clone 1 |
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Blastx results for the sequence inserted inside the nuclease CDS of survivor clone 2 |
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Blastx results for the sequence inserted inside the nuclease CDS of survivor clone 3 |
For all survivor clones, the Blastx results suggest that transposases inserted inside the sequence encoding nuclease_gp3, thereby preventing production of the nuclease and allowing bacteria to survive upon arabinose induction.
Result summary and perspective
Taken together, our results indicate that we can generate DNA-free cells thanks to the controlled expression of nuclease_gp3 in E. coli. However, resistant mutants are able to arise through the insertion of mobile genetic elements in the coding sequence. Despite these limitations, our new biobrick is a promising tool that could be used in the future for genetic containment.
