Team:UC Davis/Collaborations
CenozoicCollaboration
The 2018 Leiden IGEM team showed that nalidixic acid, an antibiotic used to treat urinary tract infections, induces cellular stress within bacteria. They hypothesized that this similar stress response might translate into mammalian systems as well.
Therefore, we collaborated with Leiden by testing four concentrations (0µM, 1µM, 5µM, 25µM and 100µM) of nalidixic acid addition in CHO DG44 cells transfected with our family of metallothionein (MT) promoters. As we described in our design page the MT1 promoter and and variations of the MT2 promoter have all been shown to activate transcription in response to cellular stresses.
Figure 1: Nalidixic acid induction of a family of metallothionein promoters. MT1 - derived from Mus musculus 305bp, MT2 full - taken from HEK293T cell line 381bp, MT2 - 59bp promoter fragment as described by Santos, Anderson K. et al[1] . Error bars represent ± one standard deviation from the mean normalized fluorescence intensity.
Figure 2: H2O2 induction of a family of metallothionein promoters. MT1 - derived from Mus musculus 305bp, MT2 full - taken from HEK293T cell line 381bp, MT2 - 59bp promoter fragment as described by Santos, Anderson K. et al [1]. Error bars represent ± one standard deviation from the mean normalized fluorescence intensity.
CHO DG44 cells were transfected with pcDNA+eGFP plasmid MT promoter derivatives using the vender supplied lipofectamine 3000 protocol. Twelve hours after transfection cells were exposed to varying nalidixic acid concentrations (0µM, 1µM, 5µM, 25µM and 100µM) and varying H2O2 concentrations (0µM, 1µM, 5µM, 25µM and 100µM) as a positive control. Cells were measured every 6 hours via fluorescence microscopy for eGFP and captured images were processed as described in our Measurement page.
Figure 1 shows nalidixic acid induces all MT promoters, albeit MT1 and MT2 most strongly. Interestingly, induction via nalidixic acid appears to be 50% stronger than H2O2, a chemical commonly used to induce cellular stress (Figure 2).
This data shows that over the concentrations we tested, nalidixic acid has the potential to cause cellular stress to both bacteria and mammals, an interesting result especially due to nalidixic acid’s pharmaceutical uses. Further testing, using additional and/or therapeutically relevant concentrations are necessary to follow up on this result.
References
[1] Santos, Anderson K. et al. "Expression System Based On An MtIIa Promoter To Produce Hpsa In Mammalian Cell Cultures". Frontiers In Microbiology, vol 7, 2016. Frontiers Media SA, doi:10.3389/fmicb.2016.01280.