Proof of Integration

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Validating APUS - Cell based experiments

Although we have shown that all the hardware within APUS is successfully integrated, the ultimate test is deriving meaningful data of a microbe from APUS. Unfortunately, we did not transform a second plasmid in time to study cell communication (See Engineering Success- Biology). Instead, 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 (Figure 1). 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 1. 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.

Improvements to the current state of the field - APUS vs. Traditional Plate Reader

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 2). 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 2. 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.

Assessing cellular communication with APUS

Looking at the monolayer chambers from which we collected data, after hour 4, cells did not divide as expected (Figure 1). Expected results were predicted to show that when constantly exposed to light, bacteria become photobleached and their fluorescence decreases. If cells are not dividing, there will be no new bacteria to replace the photobleached cells and fluorescence decreases. Although the plate reader suggests that M9 may be the better media for stronger fluorescence, APUS suggests that over long periods of time, M9 is not suitable for these cells. In fact, this may be why Chen, et. al chose LB to use in their experiments. A future experiment will be conducting an APUS experiment with LB to confirm we see stabilized fluorescence as we have seen in the plate reader. Once we successfully transform the second plasmid required for communication, we will use APUS to collect data.

Figure 3. Images of a monolayer chamber in the APUS platform over 24 hours. Images like these were captured every 5 minutes and all were analyzed with the APUS software tool to produce Figure 2. The strongest fluorescence was seen at hour 4, because cells had grown and all had high levels of fluorescence. Hour 10 shows the effect of photobleaching and how the absence of new growth prevents stable fluorescence levels. Cells began to divide again and recover their fluorescence at Hour 22.

The possibilities with APUS

An experiment like this demonstrates the power of APUS. In just one automated experiment, we can show how media impacts cell growth over long periods of time. The APUS platform gave us important data efficiently, and we would use this M9 media result to better design other experiments. This is just one example of how APUS can better characterize microbes and provide important information other than microbial communication. It shows just how versatile our platform is.