Proof of Concept

In this section we describe the proof of principle we performed to validate our system. The experiment was performed to see whether the diffusion-only design with agarose-immobilized cells in a channel (without flow) would work for the SmoColi system.

Experimental setup

Figure 3: Experimental setup for SmoColi, a tube with no flow, diffusion only

Modeling showed that diffusion and degradation of the inducer is enough to create a concentration gradient in the channel. The experimental validation of this hypothesis was first performed in a 2mm diameter tube. For proof of concept, we engineered E. coli strain JM101 to express GFP upon IPTG induction. The cells then were immobilized in agarose and the suspension was added to the tube (see Figure 4). One end of the tube was connected to a resevoir (1.5 ml) containing 10 mM IPTG solution. After incubation at 37 °C overnight, an IPTG-inducible GFP gradient could be observed (Figure 6 and 7). The experiment confirmed the modeling results. Our cells survived and we concluded that we do not need constant supply of nutrients.

Because there is no flow needed in the setup, the whole design is rather simple compared to the ones where flow is included (Microfluidic Channel Design). Here, the whole channel can simply be filled with cell-agarose suspension. Upon solidification to a gel, the channel can be connected to a reservoir containing the inducer molecule on one side. Likewise we do not need recycling because AHL can diffuse through the whole channel and does not have to diffuse against a flow. Moreover, having a tube instead of a microfluidic device would save us some time that we would need otherwise for the channel construction.

Modeling the system thus had a profound effect on the process design, leading to an extensive reduction of complexity and error-proneness. Additionally, the AHL induced alarm system would be much easier to establish in a channel without flow.

Figure 4: Photo of the channel in action. The channel (the long thin tube at the right, 2 mm diameter, 7 cm length) is physically attached to a reservoir filled with the sample medium containing the toxic molecule, or in our test system with IPTG (the Eppendorf tube at the lower left). In the case of acetaldehyde, the whole setup would be packed in an impermeable plastic bag to significantly reduce the vaporization of acetaldehyde (not shown).

Figure 5: After the experiment, the agarose gel containing the cells is removed from the tubing. The bald interior of the channel is placed on a petri dish (see picture) and analyzed under a fluorescence microscope.

Results