Tethering bacteria to a glass surface
This experiment is both simple to set up and analyze. The cells were grown, as before, in M9 medium at 30°C, which yields an average of 1.5 flagella per cell. This is crucial to tether most of the bacteria at only one flagellum. For highest motility, cells were harvested during the mid to late exponential growth phase and resuspended in a specific tethering medium. To truncate the flagella, the bacteria were pushed through a very thin needle. This is a necessary step so the bacteria bind to the antibody close to the membrane, which guarantees nice circular rotations. The microfluidic chip was prepared by incubating it with the antibody specific for the flagellar protein FliC. The antibody attaches to the glass surface, due to intermolecular forces with the glass’ silica surface. The bacteria with the sheared off flagella were then also incubated inside the channels overnight at 4°C to prevent cell division (see Microfluidic Chip).
Imaging the apartate response
The procedure described in this section was the proof-of-concept experiment for the viability of “Approach A: Chemotaxis” as a biosensor. It showed the bacteria reacting to step changes in concentration of the natural Tar ligand aspartate. The readout was acquired by imaging many bacteria with a 20x magnification phase contrast microscope. The low magnification allowed us to have enough resolution to resolve single spinning bacteria, while guaranteeing a high enough frame rate (25 frames per seconds) to also resolve the bacteria’s natural rotational frequency (2-9 Hz). As seen in (video of spinning bacteria) only about 5-10% were spinning, a value which agrees with literature . Several dilutions of aspartate ranging from 0.1 μM to 1 mM were prepared. These concentrations correspond to the natural sensing range of the Tar receptor. To expose the tethered cells to the different concentrations, the medium in the reservoir of the microfluidic chip was exchanged manually with a pipette. With the optimal flow rate of 10 μL/min (see Microfluidic Chip) the cells were induced resulting in the described change in rotational bias. We measured many different concentration changes; we increased from zero to low and high concentration and we made multiple stepwise increases or decreases.
Follow-up experiment with 𝝰-methyl-aspartate
Considering that aspartate is a natural nutrient for E. coli, we continued the response experiment with the related compound 𝝰-methyl-aspartate. This molecule has structural similarity to aspartate and is therefore also sensed by the Tar receptor. However, it is not degraded by the bacteria. Although no response was observed for very low concentrations of 𝝰-methyl-aspartate (around 1 μM), we were still able to detect a rotational bias similar to aspartate. This proves the hypothesis that other non-nutrient molecules can be sensed by the Tar receptor.
After promising result, we expanded on the idea of tethering bacteria to a glass surface by creating a flagellum, which sticks to objects, without an antibody as an intermediate molecule. This would make the setup even easier and cheaper to produce. The idea was to introduce a mutation in the flagellar gene fliC, which generates a fully functional but sticky version of the protein.[8,9,10] We generated the BioBrick “Sticky fliC” (BBa_K2845000) as a basic part as described in literature, as well as a composite part with the Sticky fliC under a constitutive promoter plus RBS (BBa_K2845001).
The readout of the chemotactic response of bacteria turns out to be a viable approach as a fast cell-based biosensor. The post-translational pathway allowed us to measure a signal seconds after induction with different aspartate concentrations. The big advantage of this approach is that it does not contain any artificial protein interactions but is purely based on the natural pathway of the Tar receptor, which has been optimized over millions of years of evolution. This promises the speed required for our biology-electronics interface.
-  Silverman, Michael, and Melvin Simon. "Flagellar rotation and the mechanism of bacterial motility." Nature 249.5452 (1974): 73.
-  Guha, Suvajyoti, et al. "Characterizing the adsorption of proteins on glass capillary surfaces using electrospray-differential mobility analysis." Langmuir 27.21 (2011): 13008-13014.
-  Block, Steven M., Jeffrey E. Segall, and Howard C. Berg. "Impulse responses in bacterial chemotaxis." Cell 31.1 (1982): 215-226.
-  Berg, Howard C., and P. M. Tedesco. "Transient response to chemotactic stimuli in Escherichia coli." Proceedings of the National Academy of Sciences 72.8 (1975): 3235-3239.
-  Larsen, Steven H., et al. "Change in direction of flagellar rotation is the basis of the chemotactic response in Escherichia coli." Nature 249.5452 (1974): 74.
-  Kuwajima, G. O. R. O. "Construction of a minimum-size functional flagellin of Escherichia coli." Journal of Bacteriology 170.7 (1988): 3305-3309.
-  Berg, Howard C., and Linda Turner. "Torque generated by the flagellar motor of Escherichia coli." Biophysical journal 65.5 (1993): 2201-2216.
-  Scharf, Birgit E., et al. "Control of direction of flagellar rotation in bacterial chemotaxis." Proceedings of the National Academy of Sciences 95.1 (1998): 201-206.