It is hardly bigger than a matchbox and yet there is a laboratory en miniature on this chip. Single bacterial cells grow in about 2000 channels of a thousandth of a millimeter in diameter and can be individually studied in detail by the researchers in Prof. Erik van Nimwegen’s group at the Biozentrum, University of Basel. By recording thousands of microscopic images at short time intervals, the precise growth and behavior of many generations of individual E. coli bacteria can be tracked over several days. The huge amount of raw data generated is automatically analyzed, and precisely quantified by new image-analysis software called MoMA, which was developed in collaboration with scientists from Prof. Gene Myers’ research group at the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden.
Microfluidic device to analyze single cell responses
“This publication is the result of more than five years of intensive collaboration with the Gene Myers laboratory. It was really a gigantic effort which caused many headaches along the way," says Erik van Nimwegen. “Using our microfluidic chip and the analysis software, we can now study precisely how genes are regulated in single cells under changing environmental conditions. In this way, we not only gain insights into gene regulatory processes but also an overview of the diversity of adaptive responses of bacteria to varying environments."
For example, it is possible to investigate how individual bacterial cells respond to a sudden exposure to an antibiotic: whether they die, stop growing, or simply continue to divide undisturbed, and how the response of a cell depends on its state at the time of exposure. In order to understand why antibiotics do not always kill all of the pathogens and why infections become chronic, it is important to understand how a single bacteria behave. But the microfluidic chip can also be used to answer other questions, such as how bacteria communicate with each other, how they respond to stress or whether the relationship of bacterial strains plays a role in adaptation strategies," emphasizes van Nimwegen. "The knowledge that we can obtain from single-cell analyses are very important, because measurements of entire cell communities are often misleading since all the heterogeneity of the the single cells has been averaged out."
Cellular memory is important for rapid adaptation
The expression and regulation of genes can be studied using fluorescent proteins. Depending on how strongly the gene of interest is activated, more or less of these fluorescent proteins are produced in the bacterium and accordingly the light intensity varies. “We have used green fluorescent protein to observe how E. coli bacteria respond to alternating nutrient changes from glucose to lactose. The corresponding regulatory system, the so-called lac operon, is the ‘hydrogen atom’ of biology. It has been studied for more than 50 years, and still, we discovered new important properties when looking at it with single cell resolution,” says van Nimwegen. “In the first round, the bacteria switched to lactose turnover with a time lag. But we figured out that these naive bacteria can react in different ways, which illuminated the underlying molecular mechanism." Repeated switching leads to a much faster adaptation of the cells as they start growing much earlier. Surprisingly, the lag times are similar in genetically related cells suggesting that bacteria retain a memory of the behavior of their ancestors.
Using the microfluidic system, the researchers have gained novel insights into the dynamics of gene regulation and the adaptation strategies of bacteria. This system is suitable for a wide range of applications. All relevant information on chip design and experiments, the MoMA software for image analysis, as well as the raw data acquired in this study are openly available online.
Matthias Kaiser, Florian Jug, Thomas Julou, Siddharth Deshpande, Thomas Pfohl, Olin Silander, Gene Myers, and Erik van Nimwegen. Monitoring single-cell gene regulation under dynamically controllable conditions with integrated microfluidics and software. Nature Communications, published online 15 January 2018
Contact: Communications, Katrin Bühler