CORDIS - EU research results
CORDIS

Biophysics in gene regulation - A genome wide approach

Project description

Biophysical insight into cell size sensing in cell division

Progression into cell cycle is controlled by various biochemical switches as well as by size. However, the precise mechanism by which cells sense size to initiate replication remains poorly understood. The EU-funded BIGGER project will study the intracellular biophysics in single-molecule detail in thousands of genetically different bacterial strains to provide mechanistic and structural insight into the bacterial cell cycle. Scientists will follow the dynamic 3D conformation of chromatin and the replication forks during the life cycle of Escherichia coli. The project's results will help clarify the role of individual gene products in replication initiation and cell division.

Objective

In this project, we will develop and use technology that combines synthetic genomics and live-cell imaging. These methods make it possible to study the intracellular biophysics at single-molecule detail in thousands of genetically different bacterial strains in parallel. Our approach is based on in situ genotyping of a barcoded strain library after phenotyping has been performed by live-cell imaging. Within the scope of the proposed project, the new technology will be used to solve mechanistic and structural questions of the bacterial cell cycle.

To this end, we will explore two parallel but complementary applications. In the first application, we will determine the dynamic 3D structure of the E. coli chromosome at 1kb resolution throughout the cell cycle. The structure determination can be seen as a live-cell version of chromatin conformation capture, where we will follow the 3D distances of 10 000 pairs of chromosomal loci over the cell cycle at high resolution. In the second application, we will make a complete CRISPRi knockdown strain library where we can follow the replication forks of the E. coli chromosome and septum formation over the cell cycle in individual cells. Using this strategy, we will resolve how individual gene products contribute to the cell-to-cell accuracy in replication initiation and cell division. In particular, this approach allows us to address the challenging question of size sensing at replication initiation. How the cell can decide that it is large enough to initiate replication is still an open question despite decades of investigations.

The general principles for high-end imaging of pool-synthesized cell libraries have nearly unlimited applications throughout cell biology. The specific applications explored in this project will take the understanding of the bacterial cell cycle to a new level and answer general questions about the chromosomal organization and cell size sensing.

Host institution

UPPSALA UNIVERSITET
Net EU contribution
€ 2 411 410,00
Address
VON KRAEMERS ALLE 4
751 05 Uppsala
Sweden

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Region
Östra Sverige Östra Mellansverige Uppsala län
Activity type
Higher or Secondary Education Establishments
Links
Total cost
€ 2 441 410,00

Beneficiaries (1)