AGILe Cell

Accessing Genetic Information in the Living Cell

In this project we will combine high-resolution single-molecule investigations in vitro with molecular dynamics (MD) simulations and experiments on sequence-specific search kinetics in living cells, in order to decipher the regulatory genetic codes.

Our understanding of the genetic code is essentially limited to the protein coding regions, for which the rules of decoding triplet nucleotides by tRNA adaptors are well known. However, hidden in the code are also instructions for how RNA and protein expression are dynamically regulated in space and time. This information is as important for life as the mechanistic function of the individual molecules. The codes are read by regulatory molecules that provide access to the correct genetic information at the right time. The average interaction patterns are often known from genome-wide, sequencing-based methods, but very little is known about how the specific genetic information is quickly and accurately searched for and retrieved when needed.

The fact that DNA has a uniform structure built from a few recurring building blocks linked into a linear sequence, facilitates its maintenance and replication. However, it also presents all macromolecules that need to retrieve genetic information with a substantial challenge; how do you find the right combination of bases in the vast pool of very similar sequences?

Cells have evolved two radically different search strategies in order to scan a genome or transcriptome in search of a specific piece of genetic code. Some proteins, e.g. transcription factors or nucleosomes, recognize a specific or preferred DNA sequence through interactions in the grooves, whilst other macromolecules are dynamically programmed by RNA molecules to recognize complementary nucleic acid sequences. Examples of the latter are Argonaute and Hfq programmed by interference RNA and small RNA, respectively, searching for complementary target mRNAs, CRISPR/Cas9 programmed by guide RNA to interrogate double-stranded DNA and RecA programmed by ssDNA searching for the other allele in homologous recombination.

The project is an interdisciplinary effort including principal investigators at Uppsala University, Stockholm University and KTH. The project is funded by a reserach grant from the Knut and Alice Wallenberg foundation.

The Knut and Alice Wallenberg Foundation

If you have general questions about the project, please contact: irmeli.barkefors@icm.uu.se

Johan Elf is Professor at Uppsala University. Together with his interdisciplinary research team, Johan is pioneering single-molecule tracking methods by means of fluorescence microscopy in living cells. Johan has focused his efforts on the study of gene regulation in bacteria, successfully combining theoretical and computational biology with experimental work and methods development to tackle challenging biological problems related to the central dogma of molecular biology.

Live-cell experiments are instrumental to measure binding and dissociation rates under relevant physiological conditions with all possible intracellular side interactions present.

Elf lab

Lynn Kamerlin is Professor at the Department of Cell and Molecular Biology, Uppsala University. She is a computational biologist using the tools of chemical physics to understand the chemical basis for complex biological and mechanistic problems.

Computational modelling will be used in the project to predict how small differences in sequence can result in chemical and structural changes that go beyond the additive contributions of individual base changes, thus making it possible for the macromolecules to distinguish the correct sequence from all others.

Kamerlin lab

Sebastian Deindl is Assistant Professor at the Department of Cell and Molecular Biology, Uppsala University. His research focuses on nucleic acid-interacting protein machines that regulate gene expression or directly alter the genetic information. By combining single-molecule fluorescence imaging techniques with structural and biochemical approaches, he aims at a molecular-level understanding of how the dynamics and structures of protein-DNA interactions.

In vitro single-molecule experiments will contribute the connection between sequences on the one hand, and the probabilities of binding at specific sequences, the distributions of dissociation rates, rates of sliding, and structural dynamics on the other.

Deindl lab

Magnus Johansson is principal investigator at the department of Cell and Molecular Biology at Uppsala University. His research is aimed at understanding molecular details and dynamics of protein synthesis in its context. To achieve this he develops new fluorescence based tools to study protein synthesis on the single-molecule level inside living E. coli cells.

Johansson lab

Professor Helene Andersson Svahn is heading the Nanobiotechnology department at the Royal Institute of Technology in Sweden and she is CEO of the startup company Picovitro AB and scientific advisor for Silex Microsystems. Her main research focus is micro- and nano-fluidic devices for biotech and medical applications.

Droplet microfluidics will be used in the project as a primary screening step to reduce the number of genetic variants from 1 000 000 to 1 0000 for in vitro and in vivo single-molecule kinetics studies.

Andersson-Svahn lab

Mats Nilsson is a professor at the Department of Biochemistry and Biophysics at Stockholm University and site director of Scilifelab Stockholm. His primary research focuses on molecular analysis with the aim of finding better methods to diagnose infectious diseases and cancer.

Mats extensive competence in molecular diagnostics will be instrumental to develop new in situ sequencing protocol to link genetic and phenotypic variation in vivo and in vitro.

Nilsson lab

Project meetings (video conferences):

  • Monday 2017-10-09, 13.00-16:00, BMC E10:3309
  • Monday 2017-11-27, 13.00-16:00, BMC E10:3309
  • Monday 2018-02-26, 13.00-16:00, BMC E10:3309
  • Monday 2018-04-16, 13.00-16:00, BMC E10:3309
  • Monday 2018-06-04, 13.00-16:00, BMC E10:3309

Project Retreat

  • Dates: January 17-18, 2018
  • Location: Stockholm area