Light & sound: a study on building blocks for bottom-up synthetic cells

This thesis is dedicated to advancing the understanding of how different building blocks and systems for synthetic cells work. Moreover, it is working on the expansion of the toolkit available to others who work on and will work on synthetic cells. Chapter 2 reviews in vitro transcription-translation with a focus on reconstituting and observing these processes in GUVs. The chapter starts with the comparison of two major approaches to synthetic biology: bottom-up, where the building blocks are combined to increase the complexity of an in vitro system, and top-down, where the complexity of an existing organism is decreased through the exclusion of non-vital elements. Then, different cell-free systems for cell-free transcription-translation, such as the PURE system and cell lysate, are discussed, along with their limitations. A summary of diverse GUV production methods is presented next, with an emphasis on the methods previously used for the encapsulation and observation of these systems. The chapter also provides a comprehensive overview of fluorescence imaging approaches for transcription and translation. Prospective ways to achieve improved transcription-translation in vitro and to solve the decoupling between these systems are presented. Particularly in the case when they are taken from different organisms, such as the PURE system, which has transcription machinery from T7 bacteriophage and translation complex from E. coli. Finally, the complexity and the demands of the synthetic cell project are discussed, along with suggestions on what it will take to accomplish it. Chapter 3 presents a technique for in vitro transcription-tracking at single-DNA-per-cell concentration. The single-molecule resolution of the DNA template and the mRNA molecules produced from it by T7 RNA polymerase was achieved by using specialized fluorescent probes — namely, intercalators for DNA and multiple molecular beacons for mRNA. The results section of this chapter showcases the sequence specificity of molecular beacons, their potent brightness increase upon activation, and their high signal-to-noise ratio, especially when the transcription was encapsulated in the GUVs. The technique was applied to compare comprehensively the multitude of commercially available T7 transcription kits based on their performance in vitro. Finally, the quantitative transcription measurement in GUVs with only a few DNA molecules each was unveiled, paving the way for further in vitro studies. Chapter 4 shifts the focus to the mechanical studies of the GUVs and cells. The quantitativ the size, density, and compressibility of the sample are then applied to develop a protocol for quantitative biomechanical measurements of these three values for cells and GUVs using the known values for the calibration sample. The sensitivity of the technique is illustrated by the measurement of empty GUVs against the GUVs containing agarose at different concentrations. Furthermore, the GUV data is presented along with density and compressibility data for two cell strains. Supplemented by complex spatial calibration of the field and a custom-written analysis script, quantitative acoustophoresis has applications spanning the fields of synthetic biology, cellular mechanics, and material science. Chapter 5 provides an outlook for this thesis as a whole.