The Elf Lab

Adventures with Single Molecules in E. coli

Lab Escape

Room Escape Stockholm, 2018

The Elf and Johansson labs rehearsed their team working skills at one of Stockholm Escape room facilities.
After an hour (or less in some cases) of intense brain activity, the teams relaxed at the rooftop bar of the Scandic hotel. Quite a pleasant outing altogether!

Single-molecule kinetics in living cells - A review

In the past decades, advances in microscopy have made it possible to study the dynamics of individual biomolecules in vitro and resolve intramolecular kinetics that would otherwise be hidden in ensemble averages. More recently, single-molecule methods have been used to image, localize and track individually labeled macromolecules in the cytoplasm of living cells, allowing investigations of intermolecular kinetics under physiologically relevant conditions. In a recent review, accepted for publication in Annual Review of Biochemistry, we illuminate the particular advantages of single-molecule techniques when studying kinetics in living cells and discuss solutions to specific challenges associated with these methods.

The image describes three general use cases for dynamic single molecule methods in living cells. (A) To determine the number of a particular molecular species and how this number varies over time, in space or between cells. (B) To measure the fractions of mobile and immobile molecules, how these fractions are distributed in the cell and whether a particular binding site is occupied or not. (C) To determine transition rates by monitoring state changes one molecule at a time.

Better Analysis of Noisy Multistate Diffusion Trajectories

Single particle tracking offers a non-invasive high-resolution probe of biomolecular reactions inside living cells. However, efficient data analysis methods that correctly account for various noise sources are needed to realize the full quantitative potential of the method. In a recent publication we report a new algorithms for hidden Markov-based analysis of single particle tracking data, which incorporate most sources of experimental noise, including heterogeneous localization errors and missing positions.

Compared to previous implementations, the algorithms offer significant speed-ups, support for a wider range of inference methods, and a simple user interface. This will enable more advanced and exploratory quantitative analysis of single particle tracking data.

Read the paper in Biophysical Journal

Figure: Statistical model selection. We generated a range of synthetic data sets with (a) three diffusive states and (b) defocus-dependent localization root-mean-square errors (RMSE), and estimated the number of states using (c) variational maximum evidence (VB) and (d) cross-validation using variational pseudo-Bayes factors (CV) and 10% of the data in the validation set.

Summer Partey!

Hagby-Focksta, 2018

With the sun shining and the BBQ smoking, the Elf and Johansson labs were enjoying each other's good company at Hagby-Focksta. Although the bottle opener was leaning towards the unorthodox and the spider mom grading the beer cellar was somewhat intimidating, everybody was having a good time. Later, when the sun set and the mosquitos joined the party, members of the Elflab learned how to master the ancient art of "rullran" fabrication over the open fire.

New analysis pipeline put to the test

Our new single molecule tracking and analysis pipeline is used for the first time in this study in collaboration with the Johansson lab.

In short, we track the movement of individual intracellular [Cy5]tRNAs by stroboscopic laser illumination with 1.5 ms laser illumination per 5 ms camera exposure frame. Combining symmetry-based spot detection and maximum a posteriori estimates of position and localization uncertainty with the u-track algorithm, we follow the movement of single molecules up to more than 100 frames. The apparent diffusion is estimated from mean-squared displacement (MSD) plots of trajectory segments.

To quantify binding events in terms of the accompanying changes in diffusion constant, we use a hidden Markov model (HMM) approach, which models the trajectories as random (Markovian) transitions between a set of discrete (hidden) states with different diffusion constants, accounts for point-wise localization errors and motion blur, and learns the underlying sequence of state transitions from data. The HMM algorithm performs maximum-likelihood inference of model parameters, and we use Akaike's information criterion (AIC) to determine the appropriate number of diffusive states to fit the data.

The analysis pipeline was validated by synthetic data based on simulated trajectories, camera noise and point spread functions. The statistics generated from the simulated data was very similar to the statistics obtained from the experimental movies and the analysis faithfully recovers the parameters of the true model (see image below). We are thus confident that the analysis pipeline is capable of producing reliable results.

The Cy5-labeled tRNAs display diffusion patterns consistent with what would be expected for a tRNA being used repeatedly by the protein synthesis machinery, i.e., random fast diffusion interrupted by immobilization events, possibly reporting on binding of the tRNAs to an mRNA-tethered ribosome.

The study is commented by Achillefs Kapanidis and Mathew Starcy in their News and Views article Tracking tRNA packages.

Left: Feature comparison of simulated [n = 16,389] to experimental [Cy5]tRNA data. Right: Estimated HMM model parameters for the simulated data set. True values from the underlying kinetic model are shown for comparison.

Searching the genome

Have you ever wondered how regulatory proteins can find their targets in the genome? Making it fast to the correct location can mean the difference between life and death in the competition for nutrients (LacI) or in the war against phages (Cas9). These two proteins have evolved different technique to speed up the search process. In this movie we get to follow the transcription factor LacI and the gene snipper Cas9 in their search for the correct nucleotide sequence. The movie was made by Visual Science.

Lab retreat, Linvallen

The Johansson - Elflab Winter Conference 2018 offered everything you could wish for in terms of scientific discussion, excellent international food, sunshine, great skiing and that occational mishap in the lift. The only thing missing was Ivan and Elias!

Happy Holidays!

Time-laps Christmas tree - Winner of the 2018 Elflab Christmas Image Award.

The DuMPLING has arrived!

The CRISPR/Cas9 and other genome editing techniques let us make genome wide modifications of DNA sequence or gene expression almost at will. At the same time our imaging technology allows exceptionally sensitive live cell phenotyping. BUT it is by combining genomics and biophysics that we could really revolutionize the way we do biological research.

We rose to the challenge by developing our culinary skills. The DuMPLING (Dynamic u-fluidics Microscopy Phenotyping of a Library before IN situ Genotyping) technique can be used to analyze dynamic phenotypes in large genetic libraries and subsequently recover the underlying genotype - all in one microfluidic chip.

As proof of principle we have targeted different components of the LacI operon in E. Coli using Cas9 silencing. From a plasmid library we express sgRNA for the different components together with a barcode that can be used to recover the genotype. The E. coli cells transformed with the plasmid pool are phenotyped one by one in a microfluidic chip allowing only one single clone to propagate in each position. When the phenotype has been determined, the barcodes are interrogated by sequential rounds of FISH probe hybridization.

In this study we only use a small library, but with this technique in place it is possible to scale up to libraries of several thousand genotypes. Effectively this means that we will be able to measure the effect of repressing or enhancing every single gene in the E. coli genome in one experiment. Adding all the different phenotypes that are possible to record using time lapse fluorescent microscopy, the DUMPLING might finally give us the tool that we need to understands how complex regulatory networks are integrated to achieve the high level of control that characterize all biological life forms.

The complete article is available in MSB.

For the greatest benefit to mankind

Johan Elf receives an award from the Swedish Foundation for Strategic Research (SSF) for his work on antiobotic susceptibility testing.

The FASTest is a microchip-based test to quickly determine antibiotic sensitivity for primarily urinary tract infections (Baltekin et al. PNAS, 2017) . "This project has a clear strategic focus in a world of ever increasing challenges caused by microbial resistance to antibiotic drugs and where the need for fast and sensitive diagnostic methods is high", according to SSF's motivation for the prize. The technique is patent-protected and is commercialized by the company Astrego Diagnostics AB, which was started by Johan Elf and his co-workers and now employs 10 people.

How do the molecular scissors find their target?

How fast can a cell locate a specific chromosomal DNA sequence specified by a single stranded oligonucleotide?

This question is relevant not only because the whole CRISPR/Cas9 system depends on this process happening reasonably fast; it also defines how efficiently a cell can locate and fix a double stranded break in its DNA and is closely related to how the Argonaut protein family can locate RNA and exert their functions in embryonic development and cell differentiation, just to give a few examples.

We have previously shown that a DNA binding protein typically finds a specific target sequence within a couple of minutes. Given that there are usually many proteins involved in the search process, this time average makes biological sense.

Image credit: KC Roeyer/ Doudna lab

In the case of CRISPR/Cas9 we expect the search time to be significantly longer. The reason for this is that the protein-nucleic acid complex does not simply rely on interactions in the DNA groves to find the target site, but actually needs to unwind the double helix and interrogate the DNA sequence to discriminate right from wrong. The search problem is somewhat reduced by the fact the potential targets are defined by the presence of a PAM sequence, but still presents a daunting task.

By following single fluorescently labeled dCas9 molecules in living E. Coli cells, we find that on average it takes the dCAS9 molecule 6 hours to find its target sequence. This number is verified by measurements in bulk where we detect Cas9 protection against cleavage by restriction enzymes. Compared to the search time of the transcription factors it's an eternity, but it is the price that the CRISPR/Cas9 system has to pay in order to be reprogrammable to target any sequence.

Whether used by bacteria as a defense against invading phages or by scientists as a method to repress a specific gene, the system requires both Cas9 and its guide RNA to be present in high concentrations in order to work efficiently.

Read the complete article in Science

fASTest: Antibiotic susceptibility testing

Antimicrobial resistance is an emerging problem globally, threatening our ability to treat common infectious diseases, resulting in prolonged illness, disability, and death. Without effective antimicrobials for prevention and treatment of infections, medical procedures such as organ transplantation, cancer chemotherapy, diabetes management and major surgery become very high risk.

Image credit: Punnoose et al. Antibiotic Resistance. JAMA. 2012;308(18):1934.

Urinary tract infections (UTIs) are among the most common types of infections, with an estimated 92 million people affected worldwide in 2013. There is widespread and strongly increasing resistance development in UTI infections. The lack of personalized treatment and pressure to reduce the use of broad spectrum antibiotics results in a significant risk of prescribing ineffective drugs.

One solution would be an antibiotic susceptibility test that is fast enough to measure the antibiotic response for every case - on site. Is this possible? What are the theoretic limits for the detection time in this case?

To meet the need for fast diagnostics of antimicrobial resistance we present fASTest. By measuring the physiological response at the level of single cells and averaging over the population the test can differentiate between resistant and susceptible bacteria within 3-11 minutes depending on the antibiotics. When tested on clinical samples, fASTest performs as good as the golden standard, which usually takes 2 days to perform. The complete study will soon be available in PNAS.

The performance of fASTest when measuring E. coli cell responds to Ampicillin (A), Amoxicillin-Clavulanate (B), Ciprofloxacin (C), Doripenem (D), Fosfomycin (E), Levofloxacin (F), Mecillinam (G), Nitrofurantoin (H) and Trimethoprim-Sulfamethoxazole (I).

Lab trip to Taipei - Taiwan 2017

The Elflab at Grand Hotel Taipei

In June 2017, the Biophysical Society arranged a thematic meeting on Single Cell Biophysics in Taipei, Taiwan. The Elflab was present in force (we missed you Prune!) and nobody regretted the 12-hour flight since the meeting was a scientific success as well as a social - well, watching David Fange and Taekjip Ha sing karaoke to Aqua's Barbie Girl was a once in a lifetime. The Elfs that stayed on for some after-the-conference-sight seeing got to experience the island's beautiful nature, close encounters with exotic animals as well as excruciating heat - at least for a Swede.

Certain Uncertainty

Super resolution means to look at things that are impossible to see. The position of the fluorophores is determined using advanced guessing and although well educated, these guesses are always more or less wrong. When super resolution data is used for down-stream analysis it can greatly improve the result to know the magnitude of these errors, i. e. to be certain about the uncertainty. This is especially true when you follow single fluorophores over time and also need to accommodate the fact the particles move in and out of focus. In a recent publication we show that pointwise localization precision can be accurately estimated directly from imaging data using the Bayesian posterior density constrained by simple microscope properties. The figure shows how the method can be used to improve detection of binding events when these are inferred from diffusion measurements of single fluorophores.

Read the complete article in Nature Communications.

The software uncertainSPT contains two basic software tools to extract and use localization uncertainty for single particle tracking and localization microscopy.

  • 1) EMCCDfit, localization algorithms to estimate particle positions and localization uncertainty from images aquired by an EMCCD camera.
  • 1b) EMCCD_dark_count_calibration.m, a calibration routine that extract EMCCD parameters from very low light images (<<1 photons/pixel).
  • 2) EMhmm, a variational EM algorithm that performs maximum likelihood inference in a diffusive hidden Markov model, where both motion blur and localization uncertainty are included in the model, through an extension of the Berglund model for single-state diffusion.
  • The code runs on matlab, with some inner loops implemented in C/C++. Binaries for 64 bit linux and max OS are included.

    Get uncertianSPT from GitHub.

Building Bridges

Johan Elf has received a VR grant of 24 million SEK over 6 years to build a research environment focusing on the physical principles for genome search by means of large scale dynamic phenotyping of strain libraries. The environment also comprises the Tenje group at the Department of Engineering Sciences, who works on microfluidic solutions for cell analysis, the Kamerlin group at the Department of Cell and Molecular biology who performs MD simulations of protein-DNA interactions and the Johansson and Deindl groups at the Department of Cell and Molecular biology that are both investigating single molecule interactions and kinetics using fluorescent microscopy.

The interdisciplinary environment will build bridges between biology, engineering and physics with many potential applications throughout the life sciences. Appealing as it may sound, collaborative efforts between engineers and scientists are rare in Sweden and much is to be gained by closing the loop between technology development and fundamental research.

If successful, the technology that we are developing will make it possible to connect dynamic phenotypes to the underlying genotype for thousands of different cell strains simultaneously. This will have important applications in many areas of science including drug discovery, biotech product optimization and basic genetic research.

MINFLUX - Resolving life

By following single molecules diffusing in the cell we can learn much about how biomolecules interact with each other in the cytoplasm. How the molecule switch between diffusive states can be used for example to estimate the reaction rate constants for molecules in equilibrium. One problem, however, has been that irreversible photo-bleaching greatly limits the individual trajectory length and thus the dynamic range of the reactions that can be studied. Historically, the only way to increase the trajectory length has been to reduce the light intensity and not collect so many photons per image, but as localization accuracy correlate negatively with the number of photons it has always been at the expense of the spatial resolution.

In collaboration with Stefan Hell's group in Goettingen we have fundamentally changed the prerequisites for single molecule tracking in living cells. Instead of imaging the light from a molecule and then use the point spread function to appreciate its spatial location, we now follow the movement of the molecule using a hollow laser beam. The fluorescent molecule is trapped in the center of the laser beam where it is not excited and thus not emitting any photons. Only when the fluorophore deviates from the middle, we detect photons that we can then use to adjust the position of the laser and bring the molecule back to the center. Since the position of the molecule is defined indirectly by the laser position, we can use the available photons much more efficiently. In principle, one detected photon is enough to know that we are out of place, and only a few more are needed to calculate a new optimal position. This means that we can record trajectory lengths that are 100 times longer than what have been possible using standard techniques. The downside is that the system is complicated to set up since laser correction must occur on the microsecond scale.

The new MINFLUX microscope will make it possible to finally study biological processes on the same spatial and temporal scale that can be reached by molecular dynamics simulations. The system that we have built in Uppsala will initially be used to study gene regulation and protein synthesis in living cells. An exciting journey into living matter at the molecular level has just begun!

Read the complete article in Science.

NOLA Elves

Scientific conferences are serious bussiness. No explanation necessary.

Light in the dark
Fluorescence microscopy has become one of the most widely used tools to study proteins in the context of the living cell, but the scientific community is still struggling with the problem of labeling proteins effectively and specifically without disturbing their natural function.

The progress in the field of live cell single molecule fluorescence microscopy that has taken place over the last couple of decades has been spectacular. However, good protocols for labeling the molecule of interest with a small, bright and photo stable fluorescent dye is limiting progress in the field, and the labeling step is where most live cell experiments fail.

We have now explored ways to use of noncannonical amino acids to introduce handles where small organic fluorophores can be bound specifically to the protein of interest. The noncannonical amino acid is encoded via the pyrrolysyl-tRNA/pyrrolysyl-RNA synthetase pair at artificially introduced TAG codons in E. coli strains that have been recoded to lack endogenous TAG codons as well as the TAG-specific release factor RF1. The amino acids contain bioorthogonal groups that can be clicked to externally supplied dyes, thus enabling protein-specific labeling in live cells with small bright fluorophores.

The incorporation of the noncannonical amino acid in the protein of interest works well. The overall labeling scheme works well works well for proteins that spend some time on the surface of the cell and thus can be clicked to the dye extracellularly. As a model protein for the NcAA-based small fluorophore labeling we use the outer membrane porin OmpC, one of the most abundant outer membrane proteins in E.coli, which is involved in the control of cellular osmolarity and the uptake of nutrients and antibiotics. The osmoporin OmpC can be labeled sufficiently specific to enable single molecule particle tracking.

In order to label proteins in the cytoplasm however, the dye has to cross the cell membrane, which proved to be surprisingly difficult. Hydrophobic dyes can cross the membrane relatively easy, but they also interact nonspecifically with various elements in the cell and thus cause high background levels. Hydrophilic dyes are less sticky but must be forced across the membrane, a process that has severe consequences on cell viability.

Read the complete article in ACS Synthetic Biology.

Homologous Recombination Explained?

A double strand break in the chromosome means trouble for any organism and the only hope resides in finding an intact copy of the break side on another chromosome. When this homologues sequence is localized, a genetic exchange takes place and both chromosomes walk away with one complete sequence. The cell is saved.

Science has presented detailed descriptions of the different steps in this homologous recombination process, all but one. According to the classical model, the broken ends randomly probe the DNA by base paring in search for the homologous sequence. If the correct site is not in absolute proximity, weeks of interrogation would be required even with a chromosome diffusing like a small molecule. Needless to say, it is not.

If the classical model is correct, homologues recombination would simply not work when the homologues sequences are not close, yet an overwhelming amount of experimental data claims that it does. We can only speculate at what might resolve this inconsistency, but a parallelized search by DNA or RNA scouts somehow produced at the break site is an attractive hypothesis. A thorough examination of available experimental observation reveals details that make this solution all the more likely. However, experimental verification of the theory is yet a challenge... Any takers?

A complete description of the hypothesis is presented in Cell systems.

Shape matters!

Have you ever wondered why DNA is a helical molecule? Although the shape of this ultimate information carrier is maybe an inevitable consequence of stacking single monomer units into a polymer, the shape also has an important effect on the time it takes for DNA binding proteins to find their targets. By solving the reaction-diffusion equations for DNA-like geometries, and complementing with simulations when necessary, we show that the helical structure can make binding to the DNA more than twice as fast compared to binding a straight molecule.

Figure: Reaction-diffusion equation and boundary conditions for example geometry. Only when the reactive patch on the sphere (gray) is in contact with the reactive patch on the cylinder do they react with rate kappa. The sphere moves around the cylinder with translational diffusion rate D. When the sphere reaches a distance Rc away from the cylinder it is equally likely to bind another DNA segment.

Check out the publication in Journal of Physics A.

Cell cycle regulation unveiled

How the cell cycle is regulated in bacteria has been examined and debated since the first model was put forth by Cooper and Helmstetter in the 1960s. Using a combination of microfluidics and image analysis of E. coli with fluorescent labels on the replication machinery, we have been able to study how the replication and division cycles are coupled in individual cells. This made it possible sort out what is the source if variability in cell sizes and generation times. In brief, all cells initiate at a constant volume per chromosome. The time from initiation to division is given by the individual cells growth rate which is variable and is the major cause of the variation in cell sizes. It has been a long project, 5 years, but it turned out very nicely in the end.

(Top) The cell volume (black solid line) of a single cell lineage simulated according to the single-cell CH model throughout an upshift in growth conditions. The growth rates are sampled from the observed distributions. When the cell passes the initiation volume(s) (red lines), a division event is triggered (green arrow) after a time period corresponding to the C and D periods (gray bars). Variation can be introduced in growth rates, initiation volume, or C+D period. (Bottom) Demonstration of how the model performance is improved as cell-to-cell variation is introduced in growth rate (mu) as well as in initiation volume (V) and C+D period (tao).

Check out the publication in Cell.

Johan Elf elected member of the Royal Swedish Academy of Science

At the General Meeting of KVA on May 11, Johan Elf was elected member of the academy's chemistry class.

Read more about the Royal Swedish Academy of Science.

SMeagol is out!

But do not worry! The latest addition to the Elfware family will not hiss in your ears when you sleep. Not unless you have a bias in your image analysis, that is ;)

SMeagol will help you optimize your experimental set-ups and make sure that there is no bias in your data analysis. The software creates realistic synthetic microscopy images by combining reaction diffusion simulations with simulations of the fluorophore photophyscis and the photophysics of the optical system. SMeagol will be presented in more detail in an upcoming issue of Bioinformatics.

(Left) Bright field image of a bacterial cell with simulated "true" locations of the molecules to be imaged and tracked. (Middle) Experimental background noise. (Right) Simulation of how the data might look like given the experimental parameters.

Download SMeagol from Sourceforge

Reaction diffusion kinetics of protein DNA interactions?
Figure: (Left) Proud supervisor (Top middle) Is that a question mark, right there? (Top right) That's some bad hat Harry! (Middle... middle) Is it only me, or does Otto look very pleased with himself? (Bottom right) The thesis committee, Prof. Lennart Nilsson, Prof. Erik Aurell and Dr. Ylva Ivarsson. (Bottom middle) The smile of a brand new doctor (and two slightly used ones). The fearsome opponent Prof. Pieter Rein ten Wolde was not so fearsome after some Champagne.

It was a few minutes to one and I would be lying if I said that the atmosphere in C8:301 was relaxed. The past few weeks had seen a desperate activity when more than four years of pioneering scientific work was to be summarized, put in context and, unfortunately, explained to the less talented. If it had only been a question of unifying the theory of micro and macroscopic interactions of proteins and DNA in a scientifically appealing manner, Anel Mahmutovic would have been home free. However, science, it had lately become painfully clear, was also about fonts, spelling and dinner arrangements…. Who would have guessed? The opponent, professor ten Woldemort, adjusted his velvet hat and made sure that his notes were in order. Finally, the clock struck one and the battle begun. G In an instance, the PhD student who trebled in the face of the fearsome professor ten Woldemort was gone, and rising from the ashes was a collected and well-spoken young scientist who met the gaze of his opponent and answered his question without the slightest tremor in his voice. A few hours later, his friends, family and colleagues congratulated Dr. Anel Mahmutovic to a job well done and a title well earned. In the evening, at first hotel Linné, it was a party wanting nothing and Anel was in every sense the perfect host he swore he’d never be.

Take a look at Anel's thesis here

Sliding - What matters?
Cartoon of the search process and the fundamental parameters involved in our model. Following dissociation the searching protein (green) can associate with rate ka to a nonspecific stretch of DNA (black) given that no other proteins (red) are in the way. Given a successful nonspecific association the protein may diffuse in 1D along the length of the DNA or perform intersegment transfer with rate kIST.

We have previously measured the sliding distance of LacI on DNA in vivo and concluded that the transcription factor uses a combination of 3D diffusion in the cytoplasm and 1D diffusion on the DNA to speed up the search for the operator. We now dig deeper into the sliding mechanism to figure out more about how the TF slides on DNA. How fast is the sliding? Is it smooth or does it include frequent microscopic jumps (hopping)? What is the influence of multiple DNA binding domains allowing the TF to transfer between DNA strands and what is the effect of molecular crowding? Using Monte Carlo Simulations scheme to realize the search process for one or two operator sites, we try to define a physically reasonable sets of parameters for which the model can explain the observed in vivo measurements.

We find that non-specific binding to DNA is improbable at first contact and that the sliding LacI protein binds at high probability when reaching the operator. We also conclude that the contribution of hopping to the overall search speed is negligible although physically unavoidable and we see an unexpectedly high 1D diffusion constant on non-specific DNA sequences. Including a mechanism of inter-segment transfer between distant DNA segments does not bring down the 1D diffusion to the expected fraction of the in vitro value, suggesting a more complicated explanation than molecular crowding.

Look out for the full article by Anel Mahmutovic that will soon be available in Nucleic Acid Research

Lab retreat, Trysil

Elflab Ski conference 2015 offered everything you could wish for in terms of scientific discussion, excellent international food, snow blizzard skiing and ambulance drivers with no sense of direction. The only thing missing was Mats, Daniel and Elias. And Cia, who spent the week-end giving birth. Big congratulations to a beautiful baby boy!

Merry Christmas and a Happy New Year

Beautiful chistmasy e. coli captured by Prune Leroy.

Nobel Prize for Super Resolution Continued

In the "Nobel studio" second episode, Swedish television presents personal portraits of the Nobel laureates in chemistry 2014 and also tries their best to explain the wonders of single molecule microscopy. We also get to share Johan Elf's view on football and we learn what popular faces might actually hide in our morning toast. But hurry, the program is only available until the 9th of January, 2015.

Nobel Prize for Super Resolution Continued

The Nobel Prize in chemistry 2014 has brought a lot of attention to the application of super resolution microscopy. Read what Åke Spross write about the research in the Elflab in UNT's Sunday edition.

How precise is cyclic life?
Figure: (Top left) The committee (Professors Mia Phillipson and Klas Hjort and Associate Professor Daniel Daley) and the respondent listens carefully as opponent Professor Peter Graumann review the process of cell division. (Top right) Mats explains... something on the whiteboard. (Bottom left) The sigh of relief as the verdict is delivered. (Bottom right) Begin the celebration!!!

How precise is cyclic life? That was the question Mats Walldén set out to answer on this cloudy December morning. After a brilliant performance from both Mats and his opponent, prof. Peter Graumann, the answer was presented; life is 20% precise. With reference to another fine piece of literature, an ultimate answer is not always as satisfying as you might think. For a more complete understanding you might read the thesis in its entirety, however, if you cannot seem to find the time, I recommend the first section life (page 11-12) and the conclusions and future outlooks (page 60-63). Below are a few lines from the introductory paragraph, a rather dystopic view of the unicellular situation. You cannot help but wondering; is there nothing we can do for these poor creatures?

"Gravity is all but suspended and instead of falling to the earth a small object is continually bombarded by its environment, forcing it to perform a random walk, to diffuse, if not attached or actively propelling itself. Inertia is negligible, so that movement stops immediately when active propulsion is discontinued. Ballistic weaponry is exchanged for a chemical arsenal as collisions carry no momentum. Sexual reproduction is rare and caring for progeny is rarer still."

Nobel Prize for Super Resolution Continued

What is the importance of super resolved fluorescence microscopy for the research in the Elflab and for our society in general, what are the life-stories of the Nobel laureates and what is the next challenge for life science microscopy? Listen to Johan Elf being interviewed by the journal Expressen.

Nobel Prize for Super Resolution

We almost take it for granted, the fact that we can peer into the nano-world and study the life and beauty of individual molecules as they go about their business in the crowded interior of the biological cell. Not so long ago this was impossible... theoretically. The theory was not wrong, but luckily there were people that refused to be held back by the seemingly impossible and eventually found a way around the diffraction limit for spatial resolution. Some of these brave scientists were awarded the Nobel Prize in Chemistry for their efforts. Some were not. We are grateful to all of you!

Nobel Prize in Chemistry 2014.

"Tracking the tracker"

After a brilliant introduction of the field by opponent Achillefs Kapanidis (Oxford) we enjoyed a scientific battle between him and Arash that lasted a good two hours (top). Afterwards the spirit remained high as Arash and co awaited the decision of the committee (bottom).

The thesis describes single-particle tracking, a technique that allows for quantitative analysis of the localization and movement of particles. Recent advances have made it possible to track hundreds of particles in an individual cell by labeling the particles of interest with photoactivatable or photoconvertible fluorescent proteins and tracking one or a few at a time. Arash show that the fluorescent protein mEos2 diffuses normally at 13 µm2/s in the E. coli cytoplasm and also that free ribosomal subunits have access to the bacterial nucleoid where fully assembled ribosomes are excluded. The thesis "Biological Insights from Single-Particle Tracking in Living Cells" can be read here.

Rivendell Seminar

Single molecules in vivo tracking reveals translation mystery

In this work, that was recently published in PNAS, we track fluorescently labeled ribosomal subunits in bacterial cells. We show that translating ribosomes move much slower than free subunits. More importantly, we show that it is only translating ribosomes that are excluded from the bacterial nucleoid, whereas free subunits have full access to the nucleoid. This finding is important because several gene-regulation mechanisms require that ribosomes are able to initiate translation as soon as the RNA polymerase has initiated transcription, and it has been difficult to reconcile such a requirement with the observation that ribosomes are nucleoid-excluded.

The intensity maps show the location of bound and free ribosomal subunits. Although the fully assembled ribosome is excluded from the nucleus, the small subunits have access and can initiate translation before the complex is translocated to the periphery.

Cruising with M/S Sjosala

The Elf lab enjoys an outing on the river Fyris. From the top, left to right: Arvid, Prune, Tom, Kalle, David, Jimmy, Ebba, Cia, Fredrik, Ozzy, Johan, Frederik, Irmeli and Mike.

Science Magazine - Editors' choice

Read what Valda Vinson write about Petter and Mats' paper "Non-equilibrium contributions to transcription factor mediated gene regulation" in Science Magazine editors' choice, Volume 343, Number 6178, Issue of 28 March 2014.

It's not that simple!

A new method for direct measurement of transcription factor dissociation makes it possible to exclude a simple operator occupancy model for gene regulation.

One by one the mysteries of gene regulation are subject to scrutiny, and our models, the way we understand gene regulation, are put to the test. We know that lacI, the repressor of the lactose digestion machinery in the cell, searches for its binding site using a combination of 3D diffusion and sliding on the DNA, we know that it slides around 45 bases before detaching and we know that it takes less than a minute for a lacI repressor to find and bind the operator, although most of the time, the TFs slide over the binding site without binding. However, to test the prevailing model for TF mediated gene regulation, which assumes that the level of repression is determined by the equilibrium binding by the repressor to its operator, we also need to know how long the TF stays bound to the DNA before it dissociates macroscopically, e.i. leaves the immediate vicinity of the regulatory site. Using a new technique based on a single molecule chase assay we can answer this question - lacI stays bound to its specific operator for on average 5 min. So far so good, with an association time of 30 s and a repression ratio of 10 (as measured by an enzymatic reporter) the model is in no immediate danger. However, if the native operator is replaced by a stronger one, this results in a slower dissociation while the association rate stays approximately the same. Conclusion; our findings do not support the simple equilibrium model and this discrepancy has to be considered when predicting gene activity from TF binding strengths. For more details, visit Nature genetics, where the study can be read in its entirety.

Prize for excellent thesis

Petter Hammar is awarded Bjurzons Premium for his thesis lac of time. The prize is awarded annually by the Uppsala University Vice-Chancellor in recognition of an excellent scientific thesis by a student or young lecturer.

What do elephants have to do with anything? Find out more in Petter's thesis

Merry Christmas

The Christmas spirit is particularly strong in the laser-lab where the new 638 nm laser takes up the competition with Rudolph the reindeer in Christmas-red glowiness.

Two x Grant

Professor Johan Elf receives funding from the European Research Council (ERC Consolidator Grant) and the Swedish Research Council (Bidrag till framstående unga forskare).

Lac sliding in E. Coli continued: Deeper into the mechanism

We have previously shown that the lac repressor slides along the DNA to speed up the search for its specific binding site. By combining micro- and macroscopic computational models, we have now been able to investigate the details of the sliding mechanism on the atomic scale. The lac dimer follows the major groove of the DNA helix, causing it to slide in a spiral motion. It remains close to the DNA for about 8bp before making a microscopic dissociation and stays on the same DNA fragment for an average 48 ms. This corresponds to an in vitro sliding length of 240 bp which correlates well with in vitro measurements. The study presents an unique combination of theoretic tools and the results satisfyingly connect macro-/mesoscopic events at the nanometer level, which enables a deeper interpretation of experimental observations.

Read more about the study here or on the PNAS web.

The making of a doctor

On a rather rainy day in June, Petter Hammar and his opponent Antoine van Oijen put on a show that kept the audience enchanted for a good 3 hours. The celebration continued at the Old Fellow House well into the following morning.

Left: Petter and Antoine deep in discussion. Top right: The committee delivers the happy news that Petter has passed the examination. From the right, Antoine van Oijen (opponent), Carolina Wählby, Yu Ji, Nora Ausmees and Johan Elf (proud supervisor). Paul Blainey and Mats Nilsson were also part of the evaluation committee. Bottom Right: The defense attracted a rather large crowd.

Rivendell Seminar: Life at low copy number

Welcome to an afternoon with excellent talks on the topic of new techniques to study the fine details of life at the molecular level:


New insights in TF kinetics

It is easy to imagine that the transcription factors (TFs) are superfast regulators of gene expression, but in reality it might take several minutes before a TF finds and binds its target sequence. As a result, negative feed-back (where the expression of a TF is inhibited by the TF itself) cannot at the same time be fast and strong.

If the TF binds strongly to the repressor site, most proteins are necessarily produced immediately after cell replication and if the binding is very week, the protein production is essentially linear; in both cases the negative-feedback is virtually nonexistent. In a recent paper in Nature Communications, we show that there is an optimal TF binding strength, where the time the binding site is free is not dependent on TF concentration. An important implication is that the TF binding strength is not necessarily correlated to its functional importance as a gene regulator.

Method Development

Single molecule tracking data usually contains large amounts of information, but extracting the data from the highly fragmented trajectories, which is often the result of SPT in vivo, can be a real challenge. Traditional methods use Mean Squared Displacement and/or Cumulative Distribution Function analysis to identify parameters in presumed underlying models, but the model has to be guessed a priori and introduction of additional states does always lead to a better fit.

In a paper that was recently published in Nature Methods, we describe an analytical tool based on a variational Bayesian treatment of hidden Markov models that combines the information from thousands of short single-molecule trajectories of intracellularly diffusing proteins. The method identifies the diffusion constants and state transition rates as well as the number of states in the model.

Using this method we have created an objective interaction map for Hfq, a protein that mediates interactions between small regulatory RNAs (green) and their mRNA targets (grey), see image to the right. Photoconvertable proteins were used to track single hfq molecules (yellow) and assign them to different kinetic states based on their diffusive properties. The diffusion constant of hfq depends on its state of binding. Free hfq diffuse fast, but when the molecule is bond to other molecules, e.g. mRNA, the molecule is slowed down. The image was featured on the cover of the March issue of Nature Methods.

Merry Christmas!

After a semester of hard endeavors the Lab Elfs take off to get some well-deserved rest...

...or will they?

New Professor!

Johan Elf is appointed Professor in Biological Physics at Uppsala University.

Lost in presumption: stochastic reactions in spatial models

During recent years, physical modeling has become increasingly important to generate insights into intracellular processes. Many times it is essential to consider both the spatial and stochastic nature of chemical reactions to be able to capture the relevant dynamics of biochemical systems. In this review, which is currently in press for Nature Methods , Fange and Mahmutovic discuss when to use, and when not to use, different models to achieve the best possible balance between speed and physical accuracy.

Different levels of quantitative modeling frameworks for intracellular chemistry.

New Release: MesoRD-1.1

MesoRD is a simulation tool developed in the Elf Lab. The software is used to simulate stochastic reaction-diffusion systems as modeled by the reaction diffusion master equation. The simulated systems are defined in the Systems Biology Markup Language with additions to define compartment geometries.

Apart from bugfixing, the new release of MesoRD updates the code for the microscopically treated bi-molecular reactions to also include reactants with different diffusion rates.

Aditional information can be found on the MesoRD web or in Fange at al. Bioinformatics 2012

Download MesoRD-1.1 here
Method Development: Gene expression analysis

Gustaf and Mats' paper "Hi-throughput gene expression analysis at the level of single proteins using a microfluidic turbidostat and automated cell tracking" is published in Philosophical Transactions B.

In order to confidently draw conclusions on the nature of transcriptional diversity it is necessary to sample a large number of cells. If done manually this could easily amount to weeks of analysis. By combining automated cell tracking with a microfluidic culture chamber this method makes it possible to analyze the rate of gene expression at the level of single proteins in a sufficient number of bacterial cells.

The movie to the right shows automated tracking of E. Coli in the microfluidic turbidostat

LacI sliding in E. coli
Petter's paper "The lac Repressor Displays Facilitated Diffusion in Living Cells" is published in Science
The lac repressor is a protein that binds to specific DNA sequences on the E. coli choromsome and regulates the activity of genes. In order to rapidly find these DNA sequenes among millions of others, we have shown that the lac repressor combines siding along the DNA sequences and free diffusion in the cytoplasm. See illustration by Tremani/Elf
Link to abstract at Science website.

MesoRD 1.0
We have released a new version of MesoRD which incorporates theory from our earlier paper, see "Approaching the molecular limit", below. The new release is found here.
Group at Ski trip to Sälen

Top row from left: Mats, Sorin, Anel, Petter, Fredrik, David and Johan.
Front row from left: Arash, Gustaf, Prune and Cia. Missing: Erik and Andreas.

Single molecule tracking in living cells
Publication: Englis et al. PNAS. We have, for the first time, been able to track individual freely diffusing proteins in living cells. This made it possible to distinguish ribosomes bound and dissociated RelA enzymes.
Approaching the molecular limit
The paper "Modeling of intracellular reaction-diffusion processes: Approaching the molecular limit" by David, Paul, Otto and Johan is accepted for publication in PNAS
Göran Gustafsson Prize
Johan Elf was awarded the 2010 Göran Gustafsson prize in molecuar biology by the Royal Academy of Sciences and the Göran Gustafsson foundation. The GG-prizes in mathematics, physics, medicine, chemistry and molecular biology are the most prestigous national scientific awards. Press info Royal Court News
Time-delayed stochastic gene regulation
The paper "The costs and constraints from time-delayed feed-back in small gene regulatory motifs" by Andreas, Per and Johan is accepted for publication in PNAS
Single molecule tracking of free proteins
The story was presented at the Biophysical Socity meeting in San Franscico. The paper "Tracking of individual freely diffusing fluorescent protein molecules in the bacterial cytoplasm" has been posted on q-Bio arXiv arXiv:1003.2110
Computational and Systems Biology
Together with the Ã…qvist, van der Spoel and Komorowski groups we have formed the new program Computational and Systems Biology
Lab-Elfs Rafting at river Fyris

New theory for search kinetics
published in Nature Physics
Move to Linneaus Center
The group is now a part of the Linneaus center for Bioinfomatics. The lab is in corridor C8:2 of the biomedical center.
The Fellowship is expanding.
Petter Hammer, Rickard Hedman and Mats Wallden are joining the lab. Welcome!
Recruitment: The Fellowship is expanding.
We have many new quests to compleat starting 2008 and 2009. Outstanding candidat es for postdoc, PhD and MS positions with a background in physic, chemistry, biology, computor science, mathematics or a mix of these are welcome to contact Johan Elf for more information.
ERC Grant

The Elf lab got the highly competitive ERC Starting Independent Research Grant. Less than 3% of the 9200 applications were granted. The grant implies that a number of postdocs and PhD students will be recruited over the next few years.

First microscope

We have started building our first single molecule microscope for live cell imaging

Ingvar Carlsson Award

J Elf was awarded the Ingvar Carlsson Award (3MSEK) by the Swedish foundation for strategic reserach.

Dr. Brian English

Dr. Brian English joins the Elflab as a postdoctoral fellow. Dr. English is an expert in single molecule sepectroscopy and got his PhD in chemistry at Harvard University.

There and back again

Returning from the Xie group at Harvard I am now starting my own group in the department of Cell an Molecular Biology at Uppsala University.

Our work will be focused at the development of new experimental and computational methods for analyzing intracellular transcription factor dynamics at high temporal resolution and spatial precision.

sample imageThe methods will be used to answer fundamental questions in bacterial physiology related to transcription factor mediated gene regulation in living bacterial cells.