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Screening candidates

Date: 2019-01-31 15:31:33.0
Author: Jon Evans

 

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In terms of producing fuels, chemicals and energy, microbes can already do a lot, and with the latest genetic engineering techniques they can be modified to do even more. But actually finding the best microbial strain for performing a particular function, whether converting plant material into a specific industrial chemical or generating electricity in a microbial fuel cell, remains a difficult, time-consuming process. Two new methods for screening microbes could help to change that.

The first method, developed by Srivatsan Raman and his colleagues at the University of Wisconsin-Madison in the US, can be utilized for determining the most effective microbial strain for producing a particular biofuel or biochemical. This method takes advantage of the fact that for a microbe to produce any biofuel or biochemical it needs to be able to generate more energy than it requires for its essential metabolism and growth. It can then utilize this excess energy to produce the desired biofuel or biochemical.

So when screening microbes able to produce a desired biofuel or biochemical, the best strain will be the one with the most excess energy available. To find this strain, Raman and his colleagues decided to develop a biosensor for detecting a biochemical called nicotinamide adenine dinucleotide (NAD). This transports electrons released from the breakdown of nutrients in a cell so they can be used to power the cell’s metabolic reactions, including those needed to produce biofuels and biochemicals. NAD does this by cycling between two different forms – NADH and NAD+ – and cells with a larger ratio of NADH to NAD+ have a greater store of energy for metabolic reactions.

The novel biosensor is based on a transcription factor, a protein that switches off a specific gene, derived from the bacterium Bacillus subtilis. Called Rex, this transcription factor is activated by binding with NADH, but its precise behavior depends on the NADH/NAD+ ratio in a cell. The idea is to insert the gene for this transcription factor into the microbes to be screened and link it to a gene for a fluorescent protein. At low NADH/NAD+ ratios, Rex will keep the gene for the fluorescent protein switched off, but at high ratios it allows the gene to switch on, causing the microbe to fluoresce.

As they report in ACS Synthetic Biology, when Raman and his colleagues tried this with Escherichia coli, they found that cells with high levels of NADH did indeed fluoresce. “What we think we have is a really simple tool that folks in the lab can use to measure the redox state of their cell type of interest,” says Raman.

In contrast, a team of US scientists led by Cullen Buie at Massachusetts Institute of Technology (MIT) in the US wanted to find a quick and easy way to identify electrogenic microbes that can transport electrons right out of the cell. Such microbes can be used in microbial fuel cells, where they generate electricity by breaking down organic material. Recent research has indicated that the ability to transport electrons out of the cell might be more widespread amongst microbes than originally thought, which a simple test would help to confirm.

To try to develop such a test, Buie and his team analyzed known electrogenic microbes with a dielectrophoresis microchip. This comprised a long channel filled with a liquid buffer; half-way along its length, this channel pinches to an aperture that is 100 times narrower. When an electric field is applied along the channel, it causes the liquid buffer to start flowing, via a force known as electro-osmosis, but the field also becomes focused at the aperture, where its strength increases by a factor of 100.

Microbes introduced into the channel are carried along by the flow until they reach the aperture, where they can become trapped by the focused electric field. When Buie and his team tried this with both electrogenic and non-electrogenic microbes, they found that electrogenic microbes could be trapped with weaker electric fields than non-electrogenic microbes. As they explain in Science Advances, they think this is because electrogenic microbes have a higher surface polarizability, meaning the degree to which charges are separated at the surface of the cell.

This is the first time a link between electrogenic microbes and surface polarizability has been detected, and potentially offers a straightforward way to identify them, especially using the dielectrophoresis microchip. “Polarizability might be something we could use as a proxy to select microorganisms with high electrochemical activity," says team member Qianru Wang at MIT.


The views represented here are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd. or of the SCI.


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