Part:BBa_M50026:Experience

All experiments were done with commercially competent NEB 5-alpha E. coli cells. Cells containing pColi, a plasmid containing genes conferring ampicillin resistance and the expression of GFP were also used. We verified the ability of pColi to drive significant GFP output in the presence of rhamnose when compared to untransformed cells. This was done by measuring the fluorescence of transformed and untransformed cells after a 24 hours growth period in media containing 1000 µM rhamnose via a fluorescence assay.

Plasmids were ordered from and synthesized by DNA 2.0. We received the plasmid in powdered form, and diluted it to a concentration of 100 ng/µL. Following a standard BioE44 Lab transformation protocol, we transformed E. coli cells with pGold. A single colony from the transformed cells was streaked onto a new LB + ampicillin plate. All experiments used cells taken from this new plate, ensuring cells have identical genetic information.

We prepared a solution of Au3+ ions by dissolving gold trichloride from Sigma-Aldrich (334049) in LB media. Our working stock of this solution was 1 mM.

Cell Viability

E. coli cells transformed with pGold were assayed for cell proliferation and growth race in various gold concentrations. For pGold cells, media was prepared at 0, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 50, 100 and 1000 µM Au3+ and ampicillin in LB stock. Absorbance readings (OD600) were measured every 30 minutes for 16 hours, and converted in to cell numbers.

Samples growing in the plate showed logistic growth patterns (Figure 1), indicating an exponential growth phase, eventually resulting in resource saturation. All samples showed rapid early growth in the first three hours, followed by a one-hour lag period. Growth rates began decreasing five hours after cells were added to the media, and most samples reached the stationary phase after eight hours. After this time, some samples remained in the stationary phase while others began experiencing a decline in cell density. This may be attributed to accumulation of toxic by-products, decreased nutrient availability, and poor growing conditions in the plate reader. In order to decouple cell viability from other factors that may have caused a decline in cell population, we focused our analysis on the early rapid growth phase.

Figure 1. Growth curves of untransformed, pGold transformed, and pColi transformed E. coli cells. All samples exhibit logistical growth, typical of bacterial cells.

Cells growing in higher concentrations of Au3+ experienced a slower growth rate three hours after cells were added (Figure 2). Cells growing in media without gold grew 363% quicker than cells growing in media with the highest concentration of gold, 1 mM. From this, it is clear that the presence of Au3+ in the form of gold trichloride negatively influences cell viability.

Figure 2. Cell growth rate from OD600 values. At three hours after cells were added to media, growth rate exhibits a negative dependence on gold concentration.

Dose-dependent response of E. coli to Au3+

E. coli cells transformed with pGold were assayed for GFP output in response to Au3+ concentrations in the growth media. Overnight liquid cell culture was used for this assay. Untransformed E. coli cells grown in LB media and transformed cells containing pColi grown in LB media with ampicillin and 1000 µM rhamnose were used as negative and positive controls, respectively. Samples were inoculated to an OD600 of 0.1 in 200 µL of media (at the same concentrations as previously described). We placed samples in an optically clear 96-well plate, continuously shaking the samples while maintaining them at 37 ˚C. Fluorescence readings were measured every 30 minutes for 16 hours at 75% gain, and samples were performed in replicates (n=4). We calculated GFP values by subtracting the fluorescence readings of blank media from sample readings and normalized by cell density. As many of the samples had high background fluorescence, we subtracted the fluorescence value of the negative control from all samples to further isolate the contribution from cell fluorescence.

GFP output data is only relevant at intermediate times around 3 hours post-inculcation of Au3+. Difficulties in interpreting data at later time points arise because of a decreased growth rate of cells after 3 hours, which is likely due to decreased viability of the transformed cells. This can be attributed to a variety of factors, including cell death, decreased proliferation due to extended exposure to gold, and depletion of essential nutrient factors in the media. From 0-2 hours, GFP expression among all test samples were indistinguishable from the negative control. This was because there was insufficient time for plasmid translation, meaning little GolS protein was formed and the GolS-Au3+ did not have time to form and meaningfully upregulate GFP expression. As such, the expression of GFP of different samples can be differentiated only at 3 hours post-addition of Au3+.

The GFP expression levels at 3 hours were normalized prior to analysis. There was insignificant induction at lower Au3+ concentrations (between 0-16 uM). Although there was slight GFP expression at these lower concentrations, possibly resulting from leaky expression of the plasmid, the GFP expression of these test samples were statistically indistinguishable from the GFP output from the negative control. It is only at concentrations of Au3+ above 32 uM that we can see strong expression levels of GFP.

Figure 3. Results of our dose-dependent GFP assay of transformed cells at 3 hours. Low induction of GFP is seen at concentrations lower than 32 uM. Concentrations at and above 32 uM up to 100 mM shows high GFP output. 1mM data is unreliable because of low cell density due to gold toxicity.

Conclusion

Past the 3 hour time point, data points for GFP output and cell density are unreliable due to gold toxicity and a host of factors affecting cell viability. However from 0-2 hours, inadequate time is allotted for the cell to express the GolS inducer, thus our test samples exhibit GFP output levels statistically indistinguishable from the negative control. It is only at the 3 hour time point that reliable data can be extrapolated for analysis. Our findings show that GFP expression is upregulated at Au3+ concentrations from 16 uM to 100 uM. The gold sensitive promoter is fully induced at 32 uM of Au3+. In addition, we found that cell viability is negatively correlated with Au3+ concentration, with the slowest cell growth occurring at 1 mM. This aligns with current literature characterizing gold toxicity, which found high gold concentrations (1 mM) resulted in massive cell death. Overall, the results are in line with the findings from the 2013 York iGEM, further validating the efficacy of the GolS inducer and GolS inducible promoter developed by the team.

Stanford Location

This plasmid is contained in E. coli in a glycerol stock at Stanford University. Name: pGold DNA 2.0 Gene: E.coli Sensor Cassette (GFP, ampicillin resistance, high ORI) Device type: Sensor Barcode Number: 0133027154 Box Label: BIOE 44 F16

References

BioE44 Protocols and Resources. “Lab: Practical #3 - Getting DNA into cells.” https://canvas.stanford.edu/courses/47957/files/folder/Labs?preview=1068554

Wang, S., Lawson, R., Ray, P. C., & Yu, H. (2011). Toxic effects of gold nanoparticles on Salmonella typhimurium bacteria. Toxicology and Industrial Health, 27(6), 547–554. https://doi.org/10.1177/0748233710393395.

York iGEM in 2013, found at this link: https://parts.igem.org/Part:BBa_K1127008.