OIn order to demonstrate the potential of our RIOT sensors to detect lactate in real-world conditions, we grew bacteria transformed with our RIOT sensors 10 and 11 in diluted supernatant of HeLa and HepG2. HeLa is a human cervical cancer cell line and HepG2 is a human liver cancer cell line. Based on Warburg effect, HeLa and HepG2 are expected to produce elevated level of lactate in the supernatant which mimics the tumour microenvironment inside our body. Therefore, if RIOT sensors are activated by lactate in the cancer cell supernatant, they have the potential to function in our body.

We measured the lactate concentration in the supernatants of HeLa and HepG2 using a lactate assay kit (Sigma-Aldrich, catalog number: MAK064), as well as the GFP intensity of transformed bacteria grown in these cell supernatants using fluorescence microscopy. We also measured lactate concentration in the supernatant of bacteria grown in LB to check whether the bacteria also produce lactate. Figure 1 is the lactate standard curve with the linear regression equation used to calculate the lactate concentration in the supernatants. As shown in Table 1, HeLa and HepG2 cells produced lactate at a concentration of 0.461 x 10^-2 M and 0.197 x 10^-2 M. In contrast, bacteria produced much lower lactate concentration.

Figure 1. Lactate standard curve.

Lactate concentration is determined by an enzymatic assay, which results in a colorimetric (570nm) product, proportional to the lactate present. Therefore the lactate concentration can be calculated based on the standards curve. This measurement was carried out using a lactate assay kit from Sigma-Aldrich.

Table 1. Lactate concentration in supernatants of HeLa, HepG2, bacteria. N = 3 ± SEM, P < 0.01

Each sample was 100x diluted. 1 μl of diluted supernatant of HeLa, HepG2 and 5 μl of diluted bacterial supernatant grown in LB and the cell media, DMEM were added into the buffer and enzyme mix separately for each well in the 96-well plate. DMEM which is free of phenol red and serum serves as a negative control.

Amount of lactate per well (nmol)= Absorbance x 0.1762

Concentration of lactate (nmol/μl) = Amount of lactate per well / Volume of sample added.

This measurement was carried out using a lactate assay kit from Sigma-Aldrich.

Figure 2. Response of RIOT sensors and other lactate sensors in the supernatant mammalian cells.

1. (A) Selected images of bacteria with fluorescence taken by microscope.
2. (B) Fluorescent images of each sensor at each condition were processed by ImageJ to obtain the corrected total cell fluorescence.

Figure 2 showed that RIOT sensor 10 (BBa_K1897028) and 11 (BBa_K1897029), as well as other sensors, were able to activate the expression of GFP under simulated conditions in the lab. Compared to the basal expression, there was a significant increase in GFP intensity, about 1.6 and 2.6 times, when RIOT sensor 10 was induced by the supernatant of HeLa and HepG2, respectively. Similarly, RIOT sensor 11 also showed a significant increase in GFP expression of about 1.9 and 1.5 times after being induced by the supernatant of HeLa and HepG2, respectively.

In conclusion, we found that HeLa and HepG2 produce elevated level of lactate and our RIOT sensors were able to detect lactate in the supernatant of these cancer cell lines. These finding supports the validity of our proof of concept that our RIOT sensors have the potential to detect high concentration of lactate in the tumour microenvironment inside the body.

Overnight cultures of bacteria transformed with different sensors were diluted. Then 40 μl of HeLa and HepG2’s supernatant were added into 160 μl of each diluted bacteria culture separately, followed by 3-4 hours incubation. If our RIOT sensors are responsive, bacteria will express GFP which can be measured by fluorescence microscope.

• RIOT sensor 10: p70-33-sfGFP-Terminator (BBa_K1897028) contains lldRO1-J23117-lldRO2 promoter (BBa_K1847008) with a weak RBS (BBa_B0033)
• RIOT sensor 11: p62-33-sfGFP-Terminator (BBa_K1897029) contains a shorter version of the promoter region for the wild-type lldPRD operon (BBa_K1897037 derived from Part BBa_K822000) with a weak RBS (BBa_B0033)
• p70-34-sfGFP-Terminator sensor contains lldRO1-J23117-lldRO2 promoter (BBa_K1847008) with a strong RBS (BBa_B0034)
• p62-34-sfGFP-Terminator sensor contains a shorter version of the promoter region for the wild-type lldPRD operon (BBa_K1897037, derived from Part BBa_K822000) with a strong RBS (BBa_B0034)