Results

After experiencing some challenges with the synthesis order, likely due to the constitutive promoter overworking the bacteria, we decided to replace it with an inducible promoter. We subsequently identified the σ38 Stationary Phase promoter designed by the IIT Madras 2008 iGEM team. This promoter is activated by the stationary-phase sigma factor and includes Lac operator sites, allowing it to be repressed by LacI unless IPTG is added to the medium. In other words, it is a dual-regulated promoter, responsive to both cellular growth phase and chemical induction. This modification was expected to reduce the stress caused by continuous transcription, potentially resolving the issue of stunted colony growth.

The same iGEM team also developed a σ32 Heat Shock promoter, which we plan to use in future experiments involving bench-scale bioreactor testing. However, for most laboratory assays, the stationary-phase promoter will serve as our primary regulatory element.

With this improvement addressing one potential limitation, we also decided to replace the reporter protein. AmilGFP, a yellow chromoprotein, proved difficult to visualize in LB medium during early qualitative assays, as its signal was barely distinguishable. To improve detection, we substituted it with mCherry, a red fluorescent protein that offers higher contrast with in the medium. With these two design changes implemented, a second synthesis order was placed for the genetic constructs shown in Figures 5–9.

After a few weeks, all synthesis orders were unfortunately canceled except for one, the construct described in Figure 8. Upon receiving this sequence from IDT, it was transformed into electrocompetent E. coli DH5α, cryopreserved with 50% glycerol, and subjected to plasmid extraction using a ZymoPURE II Plasmid Purification Kit. The extracted DNA was then analyzed via restriction digestion to verify the expected vector and insert sizes. The gel electrophoresis results are shown below: the first well contains the NEB 1 kb ladder, followed by the undigested plasmid, and lanes three and four show the NotI and XbaI + SpeI digestions, respectively.

Later, RDX detection tests were conducted using the device shown in Figure 2. This construct contains the riboswitch and amilGFP as the reporter gene, meaning that any detection of RDX could be qualitatively observed through color change. The bacteria were inoculated in LB medium with different RDX concentrations. However, the results were inconclusive because LB is naturally yellow, which interfered with the visualization of the yellow amilGFP signal.

To address this, we switched to colorless media: M9, M9 supplemented with glucose , and RPMI-1640 . Although RPMI-1640 is typically used for eukaryotic cell culture, we tested it due to its translucency. Three concentrations of RDX were evaluated in these media, and analyses were performed in triplicate for M9 with glucose and RPMI-1640.

After 24 hours of incubation, a slight yellow color was observed, suggesting potential RDX detection and amilGFP expression. Quantitative absorbance measurements at 490 nm were performed (Table 1) to validate these preliminary results. The cultures were then incubated for an additional 24 hours to determine whether the signal would intensify. After 48 hours, a stronger yellow color was visible. To better visualize this result, 2 mL bacterial pellets were generated, in which the yellow color was clearly observed. Additional absorbance measurements were also performed to confirm this observation (Table 2). Below are the images of the pellets collected for the colorimetric assay.

After 48 hours of incubation, visible color differences were observed among the bacterial pellets. In nearly all +RDX tests, at least two out of three replicates exhibited a yellow color, except for those cultured in RPMI-1640 medium. Notably, none of the –RDX controls showed any yellow color, confirming that the observed signal was specific to the RDX-treated samples. Pellets from the M9 medium displayed the most pronounced yellow color compared to their controls, suggesting stronger expression of amilGFP. Interestingly, despite this visible intensity, the corresponding absorbance readings were lower than those from RPDMI-1640 and M9+0.2% glucose media.

As shown in the bar charts in Figures 19–21, the absorbance readings for M9 medium were consistently lower than those for M9 + glucose and RPMI-1640. The combination of stronger visual pigmentation but lower absorbance may indicate a lower bacterial growth rate but higher relative reporter protein expression. These results suggest that E. coli grown in the minimal medium without a carbon source may have had greater permeability to RDX, leading to enhanced riboswitch activation and, consequently, increased reporter gene expression.

Another important observation is that the M9 control showed little to no growth, which was expected for inoculations in a minimal medium lacking a carbon source. This outcome could indicate two possibilities: (1) no cells were transferred to the control tube, resulting in no growth, or (2) the presence of RDX in the treated samples may have stimulated or supported limited bacterial growth. In either case, this experiment should be repeated to confirm reproducibility and rule out experimental anomalies. In contrast, the M9 + glucose cultures exhibited weaker yellow coloration, which could be due to reduced RDX permeability caused by the presence of glucose. However, as expected, these samples showed higher overall absorbance values, reflecting greater bacterial growth relative to the other media.

Finally, samples cultured in RPMI-1640 medium displayed no detectable yellow pigmentation across all RDX concentrations. Although this medium is nutrient-rich and optimized for eukaryotic cell culture, it was selected for testing due to its translucency and personal communication of E. coli growth in it. As shown in Figures 12–14, no pigmentation was observed in any replicate. The enriched composition of RPMI-1640 potentially interfered with RDX permeability, preventing riboswitch activation and subsequent reporter expression.