Micro-nanofabricated structures have been successfully used to study single cells by manipulating their micro-environment,10–14
but it is still challenging to realize a controllable complex fitness landscape as we seek here. Our previous experiments have been relatively simple 1-D stress experiments.7
Here, the Galapagos Islands of Darwin have been shrunk down to a 2 cm diameter interconnected network of ~ 1200 hexagonal microhabitats, each of size 200 μm diameter, which can contain up to several thousands of bacteria as shown in the . Nutrients circulate around half the perimeter of the device, while nutrients + Cirpo circulate around the other half of the device, generating gradients of the antibiotic within the array of microhabitats. The nanoslits at the periphery restrict food flow and also can generate emergent population gradients even in the absence of the antibiotic. 10 μm wide channels connecting the microhabitats allow for motion of bacteria between different metapopulations, which we will show are a critical aspect of evolution dynamics once stress resistance has evolved in a local metapopulation.
Figure 2 (A) Scheme of microecology consisting of a 2D array of metapopulation. Stable antibiotic gradient is created by flowing nutrient + ciprofloxacin containing streams in one side, while nutrient only stream in the other side. (B) Scheme by Wright5 of the (more ...)
The structure masks were designed in L-Edit and made on a Heidelberg DWL66 laser writer. There are two layers, one contains the Micro Habitat Patches (MHP), while the other contains the nanoslits. The 4” silicon wafer is first spin coated with photoresist AZ5214, then the nanoslits mask is exposed by a Karl Suss MA6 mask aligner. After developing in AZ MIF300, 100nm nanoslits are etched by Reactive Ion Etching process using a Samco 800 machine. The photoresist is removed by acetone bath. The process is then repeated with the MHP mask, but then etching on a 10 μm thickness.
Our primary choice of antibiotic to show the rapid emergence of the resistance is the important genotoxic antibiotic, Ciprofloxacin,.15
Ciprofloxacin traps the gyrase-DNA complex at the state when the DNA is cut, thereby inhibiting DNA replication and cell division,16,17
in essence preventing the cell from dividing but not killing the cell. The generation of single-stranded DNA by stalled ciprofloxacin-bound gyrase is known to trigger, via the self-cleavage of the repressor LexA, removal of LexA from transcription factor sites. Removal of LexA activates the transcription error-prone DNA polymerases.18
The effective mutagenic rate μ* due to the SOS response is 10−5
mutants/viable cell/day, 10,000 times greater than the base rate μ.19
We streaked stock E. coli stored at −80°C onto an agar plate. After overnight growth, one colony was picked and inoculated in a 15 ml tube with 3ml LB broth. After growing 3 hours, the OD reached 0.6/cm. Then 2μl of the E. coli containing medium is gently put at the inlet of center hole, without disturbing the already established antibiotic gradient gradient. Before each experiment, the chip is sealed by a glass coverslip which has spun on it a 20 μm thick elastomer PDMS gasket for sealing. The whole device is hold by an acrylic manifold with two inlets and two outlets. The medium is injected with Chemyx syringe pump. The chip is run with medium and antibiotic alone for 24 hours before inoculating bacteria to ensure a stable gradient of ciprofloxacin + nutrient and nutrient is pre-established. Time lapse fluorescence imaging is realized by Canon 5D camera using a GFP filter set (Chroma). The excitation source is a 470nm LED from Thorlabs. Images of the entire chip are taken through a Canon 65 mm macrolens by every minutes over 30 hours in an enclosure with temperature control set at 25°C.
The critical population gradients are emergent and self-generated by the bacterial chemotaxis. If the incoming food flux is much smaller than the local bacterial metabolite consumption rate in a peripheral microhabitat patch, the food gradient is self-sharpened by chemotaxis of the bacteria to the edge of the nanoslit source. However, if the incoming food flux is much larger than the bacterial metabolite consumption rate, then we have a spatially uniform food concentration. By simply varying the flow rate, the device can be operated at a desired population gradient over at least 2 orders of magnitude in steepness. Since the antibiotic is not consumed by the E. coli and there is continuous flow, the antibiotic gradient, unlike the nutrient gradient, has a constant shape once established.
An important think to note is the importance of the PDMS sealing gasket for the cover slip, which is highly oxygen permeable. The PDMS layer used for sealing the top coverslip to the chamber actually is important in oxygen transport. That is, a microhabitat with a cover slip anodically hard-bonded to the chamber resulted in no movement of the bacteria to the periphery and no evolution, presumably due to a lack of oxygen.