Statistical lower bounds on protein copy number from fluorescence expression images

Motivation: Fluorescence imaging has become a commonplace for quantitatively measuring mRNA or protein expression in cells and tissues. However, such expression data are usually relative—absolute concentrations or molecular copy numbers are typically not known. While this is satisfactory for many applications, for certain kinds of quantitative network modeling and analysis of expression noise, absolute measures of expression are necessary

Controlling and reshaping biological reaction-diffusion

Pattern formation by reaction-diffusion mechanisms is of crucial importance for the development and sustenance of all living beings. However, biological model systems so far lack the tools and versatility of the established chemical models. In this thesis, we set out to develop and expand the Min system of Escherichia coli towards becoming a universal model for biological reaction-diffusion in an in vitro setting. To this end, we firstly developed a strategy to control the Min reaction in situ. This was facilitated by incorporating a chemically synthesized azobenzene-moiety into a peptide derived from MinE. This MinE-peptide is capable of stimulating hydrolysis of ATP by MinD. Photoswitching the azobenzene crosslinker allows to also switch alpha-helicity of the peptide and therefore its activity. By periodically activating this peptide photoswitch we found resonance phenomena in the Min reaction. The photoswitch described here could thus be used in many synthetic biology scenarios, but also to learn about Min and biological reaction-diffusion systems. Secondly, we discovered that the Min system can form stationary patterns, which greatly expands the pattern diversity and therefore the phenomena which the Min model can help us understand. Especially when it comes to important decisions in development, such as cell fate or macroscopically visible effects such as fur patterns, stationary patterns are much more prominent than oscillations and waves. The discovery of these patterns also creates many opportunities for applications, especially when combined with the newly found ability of Min proteins to position arbitrary membrane-bound factors. Thirdly, this thesis shows that the Min system's complexity can be reduced even more by substituting MinE with small peptides. A combined theory-experiment approach outlines how pattern forming capabilities are restored in a small MinE-derived peptide either by adding membrane binding or by dimerizing it. This study further highlights how peptides and proteins excel as model morphogens due to their modularity and mutability. Lastly, protocols and resources are more easily available due to a combined method-paper and video that was published in open access. In conclusion, by adding tools and versatility, this thesis introduces great progress towards establishing the in vitro Min system as the ideal model for biological reaction-diffusion