Part:BBa_K4708001:Experience

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Applications of BBa_K4708001

Promoter of fluorescence and quorum sensing genes

BostonU-HW 2023

In our project, P_rhl/lac is inserted in a CFP expressing cell to study cell communication: pC165 (Figure 1). The cell with this promoter can act as a receiver cell in a quorum signaling pathway if LacI is targeted and induced by the signaling molecule (repressing the promoter's activity) (Figure 2a). The cell can also act as a sender in a quorum signaling pathway, since it promotes the expression of RhlI, producing C4HSL (Figure 2b).

Figure 1: The plasmid, pC165, in which P_rhl/lac is inserted. In pC165, P_rhl/lac drives the expression of CFP and RhlI. Kanamycin acted as the selectable marker. To turn off constitutive expression, LacI must be in the native genome or inserted via a second plasmid and induced with IPTG.

Figure 2. Two 2-pathway options within the CFP/YFP synthetic pathway. (A) Pathway 1 involves the CFP cell acting as the receiver. When activated with IPTG, the receiver will be on, constitutively fluorescing CFP. Only when the YFP sender cell produces 3-OHC14HSL (once LacI is inhibited by IPTG, driving expression of CinI) will the sender cell turn off by driving LacI expression and repressing CFP. (B) Pathway 2 involves the YFP cell acting as the receiver. The CFP sender cell will produce C4HSL via RhlI, triggering the receiver cell to produce YFP fluorescence oscillations.

We first characterized the plasmid containing P_rhl/lac, pC165, by creating a growth curve (Figure 3). The pC165 cell entered its log phase (the phase in which we want to begin experimentation) at hour 3 and growth leveled off after hour 6.

Figure 3: pC165 growth curve. We took cells from a glycerol stock and grew in LB media and kanamycin overnight. Cells were transferred to 3mL LB and kanamycin (1:1000 Kanamycin:LB) and data was recorded for 7 hours.

To strengthen our proof of concept, we chose to characterize E.coli containing Part BBa_K4708001.To test the strength of this promoter and confirm its function with the pC165 plasmid, we measured fluorescence on the plate reader (Fig. 4). The paper from which the promoter was sourced used LB as their preferred media, but we thought it promoted growing tendencies too strongly and cells would contribute less metabolic activity to fluorescence (1)(2). Therefore, we tested M9 minimal medium to see if cells fluoresced better. Compared to the CFP control, LB cells expressed more fluorescence early in the experiment. It hit its peak at hour 1.5, dropped slightly, and then stabilized. M9 cells took longer to fluoresce, but they did not drop or stabilize over the 4-hour long experiment; we expect there to be stabilization in M9, so it should happen after 4 hours. IPTG did not affect fluorescence, showing that P_rhl/lac remained active in the absence of LacI in the cell.

Figure 4: CFP fluorescence driven by P_rhl/lac from the plate reader. Data was normalized to OD and the CFP control. We took cells from a glycerol stock and grew in LB media and kanamycin overnight. Cells were refreshed in either LB or M9 media, with or without IPTG. After 3 hours of growth, 100uL (OD: 0.2) of culture was transferred to 5mL of their respective media, all without IPTG. This mimics the protocol done before APUS experiments. Data was recorded for 4 hours on a 96-well plate.

Because we couldn’t see the stabilization of M9 in the plate reader, we felt this was a good way to test APUS functionality and success compared to a standard plate reader. We measured pC165 fluorescence in M9 media with the entire APUS setup for 24 hours (Figure 5). Just as in the plate reader, there is a surge in fluorescence around hour 4, but as time progressed, fluorescence decreased. It did not stabilize as we expected for a constitutive promoter of CFP.

Figure 5: CFP fluorescence of pC165 E.coli driven by P_rhl/lac in the APUS platform. We took cells from a glycerol stock and grew in LB media and kanamycin overnight. Cells were refreshed in M9 media for 3 hours and then centrifuged into an APUS PDMS chip. Data was collected for 24 hours with cells receiving a continuous flow of M9 media (1:1000 M9: Kanamycin). 11 monolayer chambers (cell housing) were selected and analyzed with the software tool.

References: 1. Chen, Y., Kim, J. K., Hirning, A. J., Josić, K., & Bennett, M. R. (2015). Emergent genetic oscillations in a synthetic microbial consortium. Science, 349(6251), 986-989. 2. Kim, J., & Kim, K. H. (2017). Effects of minimal media vs. complex media on the metabolite profiles of Escherichia coli and Saccharomyces cerevisiae. Process Biochemistry, 57, 64-71.

UNIQ718a0a4ecf8b2ca3-partinfo-00000005-QINU UNIQ718a0a4ecf8b2ca3-partinfo-00000006-QINU