The goal of our project is to develop a self-test for colorectal cancer. A general cancer biomarker we want to sense to achieve this is lactate [1]. Some bacteria, of which Escherichia coli, contain an operon called lldPRD, which is responsible for the dissimilation of lactate. This operon can respond to lactate due to its transcription factor LldR which binds to the operon’s promoter, PlldPRD (BBa_K822000). If lactate is absent the LldR represses the operon, while if lactate is present, LldR activates expression of the operon [2]. This property has been used to produce genetic elements that are sensitive to the presence of lactate and applied as a basis for whole-cell lactate biosensors [3]. In our project we also aim to apply this to our bacterial chassis E. coli Nissle 1917 to develop a lactate sensitive biosensor.
For the proper functioning of our biosensor construct we must consider that it needs to function in a colonic environment. The lldPRD promoter does not function in the presence of glucose or the absence of oxygen. Unfortunately, both conditions are found in the human colon, meaning that PlldPRD cannot be used to sense lactate in a colonic environment [4]. Zúñiga et al. developed a lactate sensitive promoter called ALPaGA (A Lactate Promoter Operating in Glucose and Anoxia) [5]. This promoter is sensitive to lactate in the presence of glucose and absence of oxygen, perfect for developing a biosensor to sense colon cancer. Because we want to use ALPaGA to sense lactate produced by cancer cells, we set out to characterize and compare the native lldPRD operon promoter to ALPaGA. This was done by analysing both promoters in two different conditions: 1) absence of glucose, presence of oxygen and 2) presence of glucose, absence of oxygen, the results of which can be seen in Figure 1 and 2.
Figure 1: A dose-response curve for Escherichia coli Nissle 1917 grown aerobically in M9 minimal media, supplemented with 15 mM succinate and L-lactate at different concentrations. Fluorescence was measured after 16h and corrected for OD600.
Figure 2: A dose-response curve for Escherichia coli Nissle 1917 grown at microoxic conditions in M9 minimal media, supplemented with 22 mM glucose and L-lactate at different concentrations. Fluorescence was measured after 16h and corrected for OD600.
From Figure 1 can be seen that PlldPRD and ALPaGA perform similarly when glucose is absent, and oxygen is present. However, when simulating colonic conditions, meaning presence of glucose and absence of oxygen, the fluorescence output of the ALPaGA promoter is higher at all lactate concentrations compared to lldPRD.
From this data it can be concluded that ALPaGA performs similarly to the native lldPRD operon promoter in conditions without glucose and with oxygen, but greatly outperforms the native promoter when there is absence of oxygen and presence of glucose. This means that this promoter is more viable to sense lactate in colonic environments. Furthermore, it can be argued that the ALPaGA promoter more efficiently senses tumours in general by means of lactate, since the cancer microenvironment contains little oxygen but still contains glucose [6]. Therefore, we present the improved version of the lldPRD operon promoter into the iGEM registry, ALPaGA as (BBa_K4244000).
