Protein quality control is an essential process for all organisms. Despite years of research, functional redundancies and cross-talk between pathways have left many open questions about how these pathways are regulated in response to stress. In this thesis, I will present the targeted transposon sequencing approach we used to examine these pathways in strains lacking key proteases and chaperones, revealing hidden fitness determinants and interactions in the stress response. In the second chapter, I will present a computational pipeline to study gene-by-environment interactions from transposon sequencing data. Additionally, I will outline our approach to quantify the similarity of fitness profiles under different stresses to assess the effects of combinatorial stress responses. In the third chapter, I will discuss how the major chaperones and proteases obscure the correlation between transcriptomic and fitness responses. I will also explore the synthetic relationship between DNA polymerase 1 and ClpB during heat stress, as well as how heat-induced aggregation can create background-specific vulnerabilities. Lastly, in the remaining chapters, I will present intriguing results obtained during the course of this project.