During this project we have screened a whole-genome overexpression library in Escherichia coli to identify the cost of each gene. We have started with a sub-set of the whole collection, encompassing about 400 genes, and screened several different levels of expression using a robotic high-throughput setup for liquid bacterial cultures. Next, we aimed at extending the screen to the whole-genome level of 4000 genes. For that we used another robotic high-throughput setup for colony growth. The advantage is that a denser format is possible being able to screen the whole library at several, and not but a few, expression levels. This allows for the generation of high-resolution, so called, fitness functions for each gene which can be used to quantify the cost of overexpression. In the framework of this project we have found that natural regulation can result in an expression response which makes E. coli less susceptible to antibiotics. We have also found some genes, particularly from the membrane fraction, for which the exact expression level seems to be crucial to avoid detrimental effects. Those are probably under tight control and might have been optimized during the course of evolution. These results have so far been presented at several international conferences and during seminars in the systems biology community. A manuscript describing the expression-level dependent susceptibility is in preparation. Another manuscript describing overall effects of expression levels is in preparation. Both will be submitted for publication and in the event of getting published open access and visibility of the EU funding will be assured.
