Final Report Summary - DRIBAC (DNA Repair in Individual BActerial Cells)
The objective of the project is to characterize the single cell heterogeneity of DNA repair in bacteria and to evaluate its impact on cell survival. We will (i) quantify fluctuations in RecBCD protein. (ii) Manipulate fluctuation levels to measure their impact on the cell survival. (iii) Interface the experimental analysis with mathematical modelling to gain a better understanding of the system. (iv) Test the generality of the system by applying the above analysis to AddAB in B. subtilis.
We have quantified fluctuations in RecBCD transcription and translation levels by using several microscopy based assays. In particular, we have developed an in vivo single molecule detection method to count the number of RecBCD subunits in live cells. This method is based on a combination of fluorescence imaging with illumination by a laser and a microfluidic device. The experimental results have been analysed using stochastic models of protein expression.
We have shown that RecBCD subunits a present at very low levels in individual cells. Surprisingly, their level of fluctuation are lower than predicted by mathematical models suggesting than there is a specific mechanism to control them.
We have shown that our method to count proteins expressed is applicable not only to RecBCD but also to any other low expression protein that can be functionally tagged by a fluorescent protein.
We have developed several coarse grained stochastic models of double strand break repair in bacteria, which are applied to gene conversion and whole genome sequencing based data. These modelling approaches shed light on how bacteria repair their chromosomes.
Characterization of RecBCD fluctuations levels has allowed us to better understand a crucial cell process where fluctuations are expected to be substantial for physiological reasons, and of great selective consequence. Because RecBCD is also involved in acquisition of genetic diversity, the study has also shown how non-genetic heterogeneity can generate genetic diversity. In particular our understanding of RecBCD fluctuations now allows us to better understand the acquisition of new genetic information, which is often related to antibiotic resistance.