Cyanobacteria are ubiquitous autotrophic bacteria that have the natural ability to utilize sunlight to convert atmospheric CO2 into carbohydrates. Cyanobacteria’s photosynthetic abilities are very appealing to synthetic biology and biotechnology. Firstly, these organisms can be harnessed to produce compounds, such as biofuels, that they would not normally produce in the wild, but which have important economic value. Secondly, cyanobacteria possess a number of comparative advantages over land plants for this goal: their genomes are easier to manipulate, they have faster growth rates, and they can be grown in areas that are not suitable to agriculture (1). While viable biotechnological applications of cyanobacteria will ultimately require production to take place in large reactors and tanks, the optimisation of the underlying cellular process will hinge on an appropriate quantitative understanding of how biochemical networks (both endogenous and synthetic) operate intracellularly. Metabolic and regulatory networks can interact with each other in non-intuitive ways, and often the mechanisms of such interactions are only apparent at the single cell level. However, while systems and synthetic biologists have spent considerable effort in observing and building tools, such as microfluidic chemostats, to study microbes at the single cell level, these efforts have largely focused on other types of bacteria. Organisms such as E. coli and B. subtilis have been preferred because of their ubiquity as laboratory model organisms and extremely fast growth rates.
Microfluidic chemostats provide an ideal environment in which the environmental conditions can be controlled tightly. The media can be exchanged easily during the experiment thus allowing for switching between for example carbon rich and carbon poor media or media with and without specific stress inducers.
Clever microfluidics design, known as the ‘mother machine’, grows bacteria in dead ended channels with a diameter of approximately one micron (growth channels) (2). The diameter of the channels is chosen to match the average diameter of an individual rod-shaped bacterium (Figure 1).
Due to this geometry, the cells grow in a line and the cell at the bottom of the dead ended growth channel (mother cell) can, in principal, be studied for an unlimited period of time (Figure 1 A). Fresh media is supplied through a comparably wide (100 µm x 100 µm, width x depth) feeding channel. Hundreds of short (25 µm in length) growth channels are attached to the feeding channel in a 90° angle. Any bacterium that is pushed out of the growth channel as the cells divide is flushed away into the feeding channel. Fresh nutrients reach the end of channel by diffusion. The mother machine design (2, 3) has the following advantages compared to other chemostats:
1. The mother cell can be followed for an unlimited period of time.
2. As the cells grow in a line, rather than randomly orientated like in the case of a normal bacteria colony, image analysis (segmenting and single-cell tracking) is easier.
3. The media can still be easily exchanged.
This device, and variations of it, are made out of polydimethylsiloxan (PDMS) and were fabricated from a patterned silicon wafer (master) using soft lithography (2–4). The masters themselves are fabricated by photolithography (2–4). The drawback of conventional photolithography is that its resolution limit is about 1 µm thus making the fabrication of micrometre sized growth channels very challenging. An alternative to photolithography in the fabrication of such small channels is electron beam lithography (EBL). Few devices have exploited EBL to fabricate mother machines due to the high cost of electron beam time and the EBL facilities compared to conventional photolithography. However the excellent control over the pattering of feature sizes down to 10 nm and the high success rate are of great advantage A mother machine that has been produced using EBL was presented by Moolman and co-workers. They patterned a positive photoresist with EBL and etched the channels into the silicon wafer (5) (Figure 2 A). As a result the pattern on the silicon wafer was not the negative of the final PDMS device and an intermediate mold made out of PDMS had to be fabricated (Figure 2 B) before the patterns could be transferred into PDMS (Figure 2 C).
In our project we will design a microfluidic device based on the mother machine, which will be optimized for the cyanobacterium S. elongatus PCC7942 and it will allow running experiments inmultiple mother machines at the same time on one device for 7 days. Rod-shaped cyanobacteria, such as S. elongatus, can be loaded onto and grown in existing microfluidics devices (6, 7), but have a small number of specific requirements that are not always met by devices optimised for, e.g., E. coli and B. subtilis. One important difference is S. elongatus cells differ in size, being typically longer and, more importantly, wider than other common types of rod-shaped bacteria (for example, and according to our own measurements, S. elongatus cells are 50% wider on average than B. subtilis cells). These cellular dimensions require matching channel dimensions for optimal experimental performance. Another important difference is cyanobacterial cells need a light source to proliferate. Under a typical widefield microscope, the light source is attached to the condenser lens and sheds light directly above the sample at very low illumination angles. Short distances between the light source and the sample, and low angles of incidence are problematic because it is then difficult to maintain even illumination across a modestly wide field of view. Cyanobacteria grow an order of magnitude slower than E. coli and B. subtilis, and so, in order to speed up the experimental process, it would be convenient if one could test multiple strains and conditions in the same device, while keeping light conditions comparable. This goal can only therefore be achieved by building a microfluidics device with separate, but closely arranged channels that maximise space on the chip (Figure 4). To account for these requirements specific to cyanobacteria we divide the project into two main focus areas:
1. Using EBL to optimize the growth channel size to grow cyanobacteria
2. Optimization of the feeding channel layout on a device to assure even illumination on the device and increase the throughput.
PhD Student, Sainsbury Laboratory
Research Student, Department of Engineering, Academic Division: Electrical Engineering, Research group: Solid State Electronics and Nanoscale Science
Post Doctoral Researcher, The Sainsbury Laboratory
Summary of the project's achievements and future plans
Original proposal and application