Team:NUS Singapore/Proof
Proof of Concept
This page outlines the characterisation of our RIOTSensor using lactate solution. For other details, please visit the respective subpages of our wiki.
Characterisation
One of our goals is to use the RIOT sensors to detect the increased level of L-lactate produced by tumor cells in the microenvironment. To demonstrate this functional proof of concept, we measured the level of sfGFP expression under the control of lactate sensors (Figure 1). Commercially available L-lactate solution was used to activate the sensors, hence induce the expression of sfGFP. Detailed working mechanism of RIOTSensor can be found here.
The design of RIOT sensors is based on a shorter version of the promoter region for the wild-type lldPRD operon (BBa_K1897037, derived from Part BBa_K822000) and a modified version of this promoter, lldRO1-J23117-lldRO2 (BBa_K1847008), which are hereafter referred to as “p62” and “p70”, respectively for convenient purpose. We received them from team ETH_Zurich 2015. We minimise the basal expression of our reporter by linking ribosomal binding site (RBS) of different strength with these promoters (Table 1). The efficiency of the medium RBS (BBa_B0032) and the weak RBS (BBa_B0033) are about 30% and 1% relative to the strong RBS (BBa_B0034), respectively.
Table 1: Details of RIOT sensors containing a superfolded green fluorescent protein (sfGFP)
The performance of RIOT sensor 10 and 11 was compared to other lactate sensors via the fold change in GFP expression when lactate concentration increased. The results showed that RIOT sensor 10, p70-33-sfGFP-Terminator (BBa_K1897028) is more sensitive to small changes in lactate concentration (Figure 1). The p70-34-sfGFP-Terminator sensor containing lldRO1-J23117-lldRO2 promoter (BBa_K1847008) with a strong RBS and the p62-34-sfGFP-Terminator sensor containing the natural promoter with a strong RBS, had relatively high basal expression, about 7 times higher than RIOT sensor 10. In addition, they did not show significant difference in the level GFP intensity when lactate concentration increased from 10-2 M to M. Although RIOT sensor 11, p62-33-sfGFP-Terminator (BBa_K1897029) had low basal expression, it did not respond well to the increase in lactate concentration.
In general, RIOT sensor 10 not only had the lowest basal expression but also showed significant fold change in GFP intensity, which is about 2 times higher when lactate concentration increased from 10-2 M to 10-3 M.
Figure 1: Comparisons of sensitivity to changes in lactate concentration among RIOT sensors and other lactate sensors.
Overnight cultures of bacteria transformed with different sensors were diluted and incubated with various lactate concentration including 0 M, 10-3 M and 10-2 M. The best sensor is expected to have low basal expression to minimize false positive results while exhibiting sensitivity to small changes in lactate concentration.
- 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)
