In the rising field of synthetic biology (SynBio), genetic engineers build biological circuits to customize cellular processes in microorganisms. Similar to a LEGO-box, different building blocks are used in a plug-and-play kind of fashion. However, instead of using plastic bricks to create little houses, small DNA-parts are used to create synthetic circuits. They are integrated in bacteria to give them new traits in favor of humanity’s wishes. For example, bacteria can be manipulated to produce biodegradable plastics or to find novel treatments for bacterial infections. However, it is important that the circuits can function autonomously without disturbing other cellular processes. To date, the arsenal of genetic building blocks and tools that have this ability is rather limited.
That’s where bacteriophages come into play. These viruses specifically infect bacteria and can be considered as nature’s first Bioengineers. They prey on their bacterial host and hijack their metabolism in order to propagate and form new virus particles. To do this, they are equipped with numerous genetic building blocks that function independently from the bacterial host. In microbial SynBio, these phage-encoded elements can quite literally be mined to expand our ‘genetic toolbox’ for SynBio circuitry (Figure 1). This is especially necessary in non-model bacteria like Pseudomonas. They are more robust and can handle a larger variety of environmental conditions in comparison to model organisms like Escherichia coli. These characteristics enable them to thrive in more extreme environments with limited resources, which are inherent to many industrial processes. Exploiting these novel hosts might enable the transition of SynBio applications from the laboratory to the field.
In this dissertation, we’ve studied the Pseudomonas aeruginosa infecting phage LUZ100. First, we elucidated LUZ100’s infection strategy. Next, we studied the transcriptional landscape of the phage and identified multiple regulatory elements that show great potential for reprogramming Pseudomonas bacteria. To accomplish this goal, we used a state-of-the-art long-read RNA sequencing method that reveals the phage’s transcriptional landmarks and their applicability as building blocks for SynBio circuitry. By this ‘genome mining’ we’ve complemented the Pseudomonas’ genetic toolbox, which serves as the foundation of developing robust designer bacteria that can be exploited in a wide range of industrial, therapeutic and agricultural applications.
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