Paulsson, JohanGarner, EthanGutierrez Lopez, Luis Alberto2026-07-0720262026-06-052026Gutierrez Lopez, Luis Alberto. 2026. Time-Resolved Optical Pooled Screening for High-Throughput Bacterial Phenotyping. Doctoral Dissertation, Harvard University Graduate School of Arts and Sciences.32697628https://dash.harvard.edu/handle/1/42744092A central challenge in bacterial genetics is the trade-off between scale and phenotypic richness: pooled screens enable genome-wide measurements but are typically limited to bulk readouts such as fitness, whereas microscopy provides detailed morphological and growth phenotypes but has traditionally been limited to a small number of genetic variants. In this dissertation, I describe the development and initial applications of MARLIN (Multiplexed Assignment of RNA-barcoded LINeages), a method for optical pooled screening in bacteria that combines pooled genetics with multigenerational time-lapse microscopy. In Chapter 1, I describe the initial development of MARLIN in E. coli and its application to a CRISPRi screen of all essential genes. I elucidate how this platform enables the identification of phenotypes for genes of previously unknown function, reveals novel phenotypes even for extensively studied genes, and provides new insights into the stringent response, thereby illustrating its potential for biological discovery. In Chapter 2, I describe an adapted MARLIN protocol for the Gram-positive model organism B. subtilis. I then present its application to a genome-wide CRISPRi screen in B. subtilis. From these data, I report phenotypes for several genes of unknown function, identify a genetic interaction between the fla-che operon and cell division, and compare growth-length scaling between E. coli and B. subtilis. Overall, MARLIN overcomes the traditional trade-off between multigenerational time-lapse microscopy and high-throughput pooled screening. More broadly, the adapted MARLIN platform establishes a foundation for extending time-lapse optical pooled screening across diverse bacterial species.application/pdfenBacillus subtilisbacterial cell divisionbacterial phenotypingCRISPR interferenceMARLINoptical pooled screeningMicrobiologyBioengineeringCellular biologyThesis or Dissertation2026-07-070000-0001-8602-2363